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Paleoproteomics
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Cite this: Chem. Rev. 2022, 122, 16, 13401–13446
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https://doi.org/10.1021/acs.chemrev.1c00703
Published July 15, 2022

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Abstract

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Paleoproteomics, the study of ancient proteins, is a rapidly growing field at the intersection of molecular biology, paleontology, archaeology, paleoecology, and history. Paleoproteomics research leverages the longevity and diversity of proteins to explore fundamental questions about the past. While its origins predate the characterization of DNA, it was only with the advent of soft ionization mass spectrometry that the study of ancient proteins became truly feasible. Technological gains over the past 20 years have allowed increasing opportunities to better understand preservation, degradation, and recovery of the rich bioarchive of ancient proteins found in the archaeological and paleontological records. Growing from a handful of studies in the 1990s on individual highly abundant ancient proteins, paleoproteomics today is an expanding field with diverse applications ranging from the taxonomic identification of highly fragmented bones and shells and the phylogenetic resolution of extinct species to the exploration of past cuisines from dental calculus and pottery food crusts and the characterization of past diseases. More broadly, these studies have opened new doors in understanding past human–animal interactions, the reconstruction of past environments and environmental changes, the expansion of the hominin fossil record through large scale screening of nondiagnostic bone fragments, and the phylogenetic resolution of the vertebrate fossil record. Even with these advances, much of the ancient proteomic record still remains unexplored. Here we provide an overview of the history of the field, a summary of the major methods and applications currently in use, and a critical evaluation of current challenges. We conclude by looking to the future, for which innovative solutions and emerging technology will play an important role in enabling us to access the still unexplored “dark” proteome, allowing for a fuller understanding of the role ancient proteins can play in the interpretation of the past.

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1. Introduction

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The study of ancient proteins is at once a very old and a very young field. First explored in the 1930s (1) and later formulated as “paleobiochemistry” in the 1950s, (2) the early history of ancient protein research is deeply rooted in the fields of chemistry, anthropology, and geology. However, it was only following the application of soft ionization mass spectrometry in the early 2000s (3) that the study of ancient protein sequences became truly feasible, developing into the field known as paleoproteomics today.
Ancient protein research is now advancing at a rapid pace, and its application includes the study of a wide range of archaeological, historical, and paleontological remains and materials. (4−7) Often compared to its sister field of paleogenomics, paleoproteomics is not yet as developed in scale or scope, but its demonstrated success in retrieving biomolecular sequence data from samples beyond the limit of ancient DNA (aDNA) and its ability to characterize specific tissues and biological processes make it particularly valuable and give it enhanced interpretive nuance.
To probe past life using biomolecules, in the time scale of millions of years, proteins are likely to be our best resource. Proteins are found in almost all biological tissues, and before the age of plastics they also made up a large proportion of the material culture produced by human societies around the world. Proteins persist long beyond their biological function, becoming foods, textiles, building materials, paints, and glues. The remnants of these past materials and activities have become incorporated into the historical and archaeological records, just as the remains of humans, animals, and plants have become integrated into the bio- and geosphere, where they can remain accessible into deep time.
Although proteins decay, nitrogen recycling is not completely efficient, and in protected environments (e.g., bones, teeth, eggshell) proteins can persist for millions of years or more. Protein fragments are recognizable in fossils (e.g., seeds, bone), worked biological remains, (e.g., wood, textiles, archaeological and art historical artifacts), as residues on cooking vessels, and also entrapped within soils and sediments. There is more protein nitrogen in this “dead pool” than there is in all the living cells on earth. (8) Encoded by DNA, proteins pack the same amount of sequence information into approximately one-sixth the number of atoms. For example, a 50 bp fragment of DNA (30.4 kDa) has a larger mass than many intact proteins, including β-lactoglobulin (18.4 kDa), hemoglobin (15.9 kDa), and amelogenin (24.1 kDa). Protein folding and aggregation further protect proteins from chemical attack and facilitate entrapment. With fewer atoms, fewer chemical bonds, and a more compact structure, proteins consequently fall apart more slowly than DNA. However, the greater range of reactive species and our limited ability to recover direct information about their state of decay mean that ancient proteins stretch the limits of our understanding of decay processes and diagenetic modification. Yet the results are hardly esoteric, as modifications associated with ancient proteins have relevance for understanding aging and diseased tissues, and are induced during the production and consumption of protein-containing materials and foods.
In this review, we discuss the history of paleoproteomics, the revolutionary change brought about by mass spectrometry, and the methods and applications currently in use. We further detail the main challenges facing ancient protein research today and offer perspectives on future directions in the field.

1.1. Proteins as a Bioarchive of the Past

Proteins are long-lived biomolecules capable of surviving over millions of years. (9,10) They routinely outlast even the oldest surviving DNA, (11−14) and their full longevity has yet to be determined. (15−17) Although proteins do not persist into deep time as long as lipids, (18) their sequence diversity makes them more informative, and consequently proteins represent one of our most valuable bioarchives of the past.
The longevity and biological utility of proteins derive in large part from their structure. Proteins are large biomolecules built from linear sequences of amino acids folded into complex three-dimensional forms. The 20 standard amino acids, each formed around a central carbon, contain a carboxyl group and an amino group, which form the peptide bonds linking the amino acids together into proteins, and an R group, which varies between amino acids and imparts distinct chemical properties. R groups are chemically diverse, consisting of positively charged, negatively charged, polar, and nonpolar groups that can be small, large, or structurally constrained. The sequence of amino acids making up the primary protein structure is encoded by DNA, which is then transcribed to RNA and translated into proteins using trinucleotide codon sequences for each amino acid. Because proteins are derived from the genetic code, individual proteins preserve part of the heritable genetic signal of an organism, and therefore, protein sequences can be used to make taxonomic identifications and reconstruct phylogenies. (19−21)
After protein synthesis, additional post-translational modifications (PTMs) can be made to the amino acids, changing their chemical properties. (22,23) Protein splicing, autoprocessing, conjugation, and other forms of modification further expand the biochemical complexity of proteins. (24,25) This biochemical diversity makes proteins substantially more complex than other biomolecules, such as lipids or DNA, and it drives the folding of the linear primary amino acid chains into more complex secondary, tertiary, and quaternary structures, which form the basis of the diverse structural and functional roles of proteins.
During life, proteins are regularly degraded after their functional or structural roles are complete in order to recycle the amino acids for the creation of new proteins. While the average protein lifespan for mammals is only 1–2 days, (26) the longevity of a specific protein sequence ranges from minutes (e.g., transcription factors (27) and immune ligands (28)) to the entire lifetime of the organism (e.g., enamel (29) and eye lens proteins (30)). In addition, secreted proteins, such as hair keratins and silk proteins, form the basis of nonliving tissues and structures that can persist for centuries or more after the death of the organism. (31,32) Although proteins contain less genetic information than DNA (due to both codon redundancy and the absence of noncoding sequences), they are typically orders of magnitude more abundant, with many copies of a protein being made for every genome. Moreover, the tissue-specific expression of some proteins provides additional information about a given sample (e.g., milk vs muscle; leaf vs seed) that cannot be obtained from the genome alone.
The goal of paleoproteomics is to recover, identify, and study these proteins long after their natural lifespan, and typically after they have been extensively modified by taphonomic forces over centuries, millennia, or even millions of years as they transition from the biosphere to the lithosphere. The longevity of proteins, coupled with their biological ubiquity and diversity, makes them ideal subjects for exploring the deep and recent past, and as such they represent one of our most powerful tools for reconstructing biological and cultural history.

1.2. Origins of Paleoproteomics

The idea of using proteins to study deep time is not new. Almost 20 years before the discovery of the structure of DNA (33) and the formulation of the theoretical framework of the Central Dogma that defines the relationships between DNA, RNA, and proteins, (34,35) chemists were trying to use antisera to detect proteins in mummies and skeletons for the purpose of anthropological blood typing. (1,36,37) Interest in ancient proteins fell away during the war years, but was revitalized in the 1950s by geophysicists working in government laboratories whose interests had shifted from bomb-making to deep time “paleobiochemistry”. During the 1970s and 1980s, interest in immunological assays returned, followed by attempts to sequence ancient proteins using Edman degradation in the 1990s and eventually mass spectrometry during the 21st century (Table 1).
Table 1. Comparison of Instruments and Approaches Used in Ancient Protein Studies
 Immunological assaysEdman sequencingMALDI-TOFMALDI-TOF/TOFLC–MS/MS
First use on ancient proteins1937, (1),a 1980, (608),b 1984 (58),c1990 (65)2000 (3)2005 (74)2006, (89),d 2011 (91),e
Good for complex samples?YESNOTo some extentTo some extentYES
Good for samples without reliable composition?To some extentNONONOYES
Can get sequence data?NOYESNOYESYES
Can target specific proteinsYESNONONOTo some extent
Proteins detected in one analysis1–511–201–20100+
Feasibility for ancient samples++++++++++++++++++
Reproducibility+++++++++++++
Relative price per sample$$-$$$$$$$$$$$$$$$$f
Analysis time per sample++++++++++++++++++
Sample types analyzedAny sample typeSingle peptidesSample with a few dominant proteinsSample with a few dominant proteinsAny sample type
ExamplesHemoglobin, albumin, pathogens, silkOsteocalcinCollagen, keratins, silk, shellCollagen, keratins, silk, shellProteomes of bone, enamel, dental calculus, artist materials
a

Use of antisera.

b

Use of radioimmunoassay.

c

Use of ELISA.

d

Use of LC–MS/MS to identify individual ancient proteins.

e

Use of LC–MS/MS to characterize an ancient proteome of >100 proteins.

f

Depends on immunoassay design and whether antibodies are commercially available.

1.2.1. Paleobiochemistry

It is rare for any discipline to have its genesis at a single institution, yet Phil Abelson (Box 1) and the members of the Geophysical Laboratory at the Carnegie Institution of Washington, which he led from 1953 to 1971, accomplished just that. In the postwar years, they were “free to do fundamental research unhampered by the pressures that attend work in industry and government or by the teaching load that often handicaps the university scholar”, (38) and their time was heavily invested in pioneering the study of amino acids and proteins in deep time. The publication of Abelson’s article “Paleobiochemistry: organic constituents of fossils” (2) is generally considered to mark the beginning of ancient protein studies, and in just over 1000 words he outlines a vision for what has become the field of paleoproteomics. Innovations in the detection, separation, and quantification of amino acids by members of the Geophysical Laboratory, as well as the characterization of their chirality and isotopic abundance, drove research to explore ancient proteins and their mechanisms of survival, decay, and isotopic fractionation over the next two decades. Much of this early research is detailed in the book Biogeochemistry of Amino Acids. (39)
Box 1
Early Pioneers in Paleoproteomics
Phil Abelson, who headed the Geophysical Laboratory at the Carnegie Institute, Washington, DC., from 1953 to 1971 was the first to identify amino acids in invertebrate and vertebrate fossils. A nuclear physicist who worked on the Manhattan Project, his pioneering work on the “paleobiochemistry” of Oligocene clams and other fossils in the 1950s (2,581−583) marks the beginning of ancient protein studies. Ed Hare spent almost all of his career in the Geophysical Laboratory and wrote one of the earliest Ph.D. theses on the subject of ancient proteins, focusing on the amino acid profiles of modern and fossil mussels. (584) His specialty was the chirality of fossil amino acids, (40,585−588) which led to the development of the chronometric technique of amino acid racemization dating. (589−591)Tom Hoering was the third member of the pioneering team based in the Geophysical Laboratory. A chemist, he specialized in mass spectroscopy and was an early pioneer of isotopic studies of organic matter (592,593) but also conducted key early experiments into the diagenesis of proteins. (594) A major impact of the trio of Abelson, Hare, and Hoering was the talent that they drew to the laboratory, who then shaped the study of amino acids and isotopes in the coming decades by scientists including Richard Mitterer, Marylin Fogel, Noreen Tuross, Mike Engle, John Wehmiller, Giff Miller, Steve Macko, Glenn Goodfriend and John Hedges.
Ralph Wyckoff was the first to seriously study amino acid decay and contamination in fossils. A skilled chemist and microscopist, Wyckoff began his career in the field of X-ray crystallography and later contributed to electron microscopy and vaccine development. He showed that while Pleistocene bones in the La Brea tar pits retained clear microscopic evidence of collagen fibers, their amino acid profiles were highly variable and differed from that of collagen, (50,51) a finding he attributed to protein degradation and amino acid instability. He found similar results for dinosaur bones (53) and mollusc shells, (52) suggesting that fossil amino acid profiles were generally unsuitable for inferring phylogeny.
Peter Wesbroek, a geologist from The Netherlands, was influenced by the work of Margaret Jope, (595) with whom he trained in biochemistry. After returning to The Netherlands, he established the Geobiochemistry workgroup at the University of Leiden. He studied the process of biomineralization and in doing so pioneered the use of antibodies to study ancient shell (and bone) proteins (55,596,597) as part of a wider effort to understand the role of biological systems in geological processes. (598)
Peggy Ostrom pioneered the use of mass spectrometry in ancient protein studies, innovating new methods in organic geochemistry, including stable isotope-based reconstruction of paleoecologies (599,600) and the use of isotopic analysis to identify diagenesis and contamination in fossils. (601,602) Later, she was the first to make major gains in determining ancient protein sequences by applying a variety of mass spectrometry techniques, including peptide mass fingerprinting, (3) postsource decay sequencing, (70) and tandem mass spectrometry, (69) to the study of bone osteocalcin.
One of the major outcomes of this early phase of work was the development of amino acid racemization (AAR) geochronology as a tool for the comparative dating of fossils, (40) a project initiated by Abelson and his protege Ed Hare (Box 1). However, this method, which examines the chiral conversion of l-amino acids into d-amino acids (either peptide-bound or free), (41) became mired in controversy following the radiocarbon redating (42) of skeletons previously analyzed by AAR that had placed the arrival of humans in North America before the last glacial maximum. (43) Although subsequent taphonomic and technical challenges further slowed the development of AAR as a relative dating technique, (44,45) recent methodological improvements and an improved understanding of biomineral diagenesis are leading to a renewed interest in the approach, (46,47) which shows particular promise for dating materials that are low in organic matter, are subject to large 14C reservoir effects, or are beyond the limit of radiocarbon dating. (46,48,49)

1.2.2. Diagenesis, Contamination, and a Return to Immunology

Despite the early successes of the Geophysical Laboratory in recovering amino acids from fossils for the study of chemical evolution, work by Ralph Wyckoff (Box 1) and others in the 1960s made it clear that fossil proteins were highly degraded and exhibited altered amino acid profiles. (50−54) As such, they were generally unsuitable for inferring phylogeny. The absence of measurable hydroxyproline in most dinosaur bones also led Wyckoff to question the origin of the fossil-derived proteins and whether they could have resulted from the recent activity of soil microorganisms. (53)
In light of the diagenetic variability of fossil amino acids, and lacking the ability to sequence proteins directly, researchers in the 1970s and 1980s returned to immunological techniques. In 1974, Elisabeth de Vrind-de Jong (nee de Jong), Peter Westbroek and colleagues used antibodies to detect the apparent survival of epitopes in 70 Ma fossil cephalopods by immunodiffusion, (55) and Jerry Lowenstein and colleagues investigated the immunological similarity of ancient mammoths, bison, and humans to modern elephants, cattle, and humans, respectively. (56) They were able to correctly infer the systematic position of the mammoth and Tasmanian wolf using radioimmunoassay (RIA) techniques. (57) In an attempt to yield more meaningful immunological results for taxonomic systematics, an enzyme-linked immunosorbent assay (ELISA) was developed for fossil shells, (58) then later for fossil bones and teeth, (59,60) but results were difficult to replicate, and by the 1990s and 2000s immunological approaches became increasingly dogged by concerns about contamination and cross-reactivity, (61−63) as well as a lack of understanding regarding which proteins were being immunologically detected (see also ref (64)).

1.2.3. Protein Sequencing

The first successful recovery of an ancient protein sequence was made by Lila Huq (65) using Edman degradation sequencing of osteocalcin from the bone of a moa (Pachyornis elephantopus), an extinct flightless bird. This achievement was particularly remarkable, as one of the authors of this review (M.C.) will testify, because this technology is extremely ill suited to ancient proteins. Because only a single peptide can be sequenced at a time, proteins must be isolated, digested, and purified in sufficient quantity (100 pmol) prior to analysis. Moreover, the sequencing reaction will not initiate if the reactive amino terminus is modified (e.g., by pyroglutamate), and derivatization stalls in the presence of non-α-amino acids (such as isoaspartic acid, the primary product of asparagine deamidation and aspartic acid racemization). In addition, because yields fall with each successive hour-long derivation cycle, the method is slow and limited to sequences of approximately 30–50 amino acids with high accuracy. (66,67) Even today, Edman sequencing is expensive and laborious, requiring a full day or more to sequence a single peptide using automated instruments (Table 1), although massively parallel sequencing is now being developed as a tool for single molecule sequencing. (68) Since its initial demonstration, Edman sequencing has been rarely applied to ancient proteins, but was notably used to confirm the sequence of an osteocalcin peptide identified in an early application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) from a 42 ka horse bone. (69) In the wake of the frustration surrounding Edman degradation, it was Peggy Ostrom and colleagues (3) who made the first major breakthrough in recovering ancient protein sequences by successfully applying soft-ionization mass spectrometry to ancient proteins for the first time (Figure 1).

Figure 1

Figure 1. Milestones in ancient protein mass spectrometry. The broadest applications of protein mass spectrometry in archaeology today are ZooMS (zooarchaeology by mass spectrometry), which applies MALDI-TOF MS peptide mass fingerprinting to collagens, keratins, and other high abundance proteins (left), and shotgun proteomics, which uses high-resolution LC–MS/MS to identify diverse, low abundance proteins in complex mixtures (right).

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1.2.4. Mass Spectrometry Revolution

To this day, it seems remarkable that Peggy Ostrom’s breakthrough mass spectrometry work (3,70) was not deemed sufficiently significant to be published in any of the highest impact journals, even with the support of Geoff Eglington, the “father of organic geochemistry”. Her landmark studies combined gel and immunoassay work with MALDI-TOF peptide mass fingerprinting (PMF) and postsource decay (PSD) sequencing to conclusively demonstrate the presence of osteocalcin in bison bone and its survival into the late Pleistocene. This combination of immunological and mass spectrometric tools for the detection of ancient proteins, also applied by Mary Schweitzer and colleagues on mammoth bones, (64) is notable not only for the prospect of triangulating evidence but as marking the major point of transition between the two methods. While mass spectrometry has subsequently become the go-to tool for ancient protein studies, the use of immunological approaches have waned. Nevertheless, given the strengths and weaknesses of each method, there remains scope for future integration, particularly with the potential of immunoaffinity chromatography to target and enrich (or deplete) specific proteins prior to sequencing by mass spectrometry. (71)
The first widespread adoption of mass spectrometry within archaeology came with the use of MALDI-TOF MS to aid in the taxonomic discrimination of animal bones, initially sheep and goat, based on collagen peptide mass fingerprints. (72,73) While earlier efforts to characterize fossil proteins using MALDI-TOF of intact purified osteocalcin (3,69,70,74) had ultimately proven impractical due to protein degradation, (75) the application of MALDI-TOF to collagenase-digested (76) and later trypsin-digested bone collagen (72) proved a major breakthrough. This resulted in the development of the powerful PMF technique known as zooarchaeology by mass spectrometry, (6,7,77) which was given the acronym ZooMS to highlight the speed of the method and its roots in both zooarchaeology and mass spectrometry. (78) The low cost of the method and its suitability for high throughput sample processing has made it particularly powerful for many applications in archaeology, ecology, and cultural heritage (for a review see (7)), and major advances have been made over the past decade to expand the number of ZooMS markers to include a wide range of both terrestrial (79) and aquatic (80) mammals, as well as fish, (81) birds, (82) and reptiles (83) (Figure 1). Similar PMF-based approaches are also in development for additional proteins beyond collagen, including keratins (84,85) and matrix proteins in eggshell (86,87) and mollusc shell. (88)
Beyond postsource decay (PSD), (70) the use of true tandem mass spectrometry (MS/MS) to achieve a more accurate determination of peptide sequences was first achieved by John Asara and colleagues on mammoth bone using LCQ quadrupole ion trap MS/MS. (64) This was followed by the characterization of Neanderthal osteocalcin by Christina Nielsen-Marsh and colleagues using MALDI-TOF/TOF (74) and of egg proteins in Renaissance paintings by Caroline Tokarski and colleagues using nanoLC/nanoESI/Qq-TOF MS/MS. (89) These early applications of MS/MS utilized a wide variety of instrument setups, ionization techniques, and detectors, but current MS/MS analyses of ancient proteins primarily rely on LC–MS/MS systems integrating UHPLC, nano-ESI, and typically an Orbitrap (90) high performance hybrid mass spectrometer. Enhancements in speed, and more significantly for ancient samples, resolution and mass accuracy, have increased the numbers of acquired spectra and improved the success of matching these spectra to peptides. Further emerging techniques for improved ion separation, such as ion mobility, also show great promise for improving data acquisition. However, ancient proteomes typically contain many fewer proteins, and with greater levels of modification, than equivalent modern samples. Therefore, the discipline, while still in its infancy, is currently more stifled by downstream analysis than instrumental limitations.
The major strength of MS/MS is its capacity for analyzing complex protein mixtures. The use of MS2 spectra to determine the sequence of peptides from degraded whole proteomes (91) and its integration with genomics (92) represented the next steps in the maturation of the field, followed by applications in phylogenetic interpretation, (20,21,93) sex determination, (94) food preparation, (95) pathology, (96) art history, (97,98) and residue analysis, (99) among others. Major milestones in the development of mass spectrometry techniques and applications for the study of ancient proteins are highlighted in Figure 1.

2. Ancient Proteins

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Today, paleoproteomics is a dynamic, fast-paced, and growing field. Regardless of the analytical techniques applied, all ancient protein studies share certain challenges and must (1) consider the formation, incorporation, and degradation processes that precede the recovery of ancient proteins, (2) apply methods to extract and prepare proteins for analysis, and (3) select appropriate analytical and interpretive strategies for characterizing ancient proteins (Figure 2). During each stage, there is progressive loss of the original proteome, an increase in chemical complexity due to diagenesis, and the addition of contaminants. Choices made in instrumentation, database selection, and data processing steps can have large impacts on the reconstruction and interpretation of ancient proteomes.

Figure 2

Figure 2. Conceptual stages of protein incorporation and recovery in archaeological samples. Archaeological proteins represent a small fraction of the proteins that were once present during life. Careful consideration of the full history of a sample, from incorporation to analysis, must be taken into account in order to make accurate inferences about the past.

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2.1. Pathways of Incorporation

Understanding the manner by which a protein was formed and how it was incorporated into a given sample is the first step in ancient protein analysis. For some samples, the manner of protein incorporation is obvious, as is the case for endogenous proteins in proteinaceous tissues such as collagens in skin and bone, keratins in hair and feathers, amelogenin in tooth enamel, and matrix proteins in mollusc shells. In such cases, the proteins comprise the tissue itself and were incorporated at the time of tissue formation. Nevertheless, some processes, such as biomineralization, are extremely complex and remain incompletely understood. (100) During biomineralization, other coassociated endogenous proteins, such as blood and plasma proteins in bone, may also become incorporated into the tissue, but the extent of biological variability related to such protein incorporation is not well studied for many tissues.
In other cases, the manner of incorporation may be less direct. Dental calculus, for example, is a calcified microbial biofilm, but in addition to bacterial proteins the dental calculus proteome is also rich in human digestive enzymes (e.g., salivary α-amylase) and immune proteins (e.g., α-s1-antitrypsin, myeloperoxidase, neutrophil defensin) that originate from saliva and gingival crevicular fluid, respectively. (92) The proteins of these fluids, which continuously bathe the teeth, become incorporated into dental calculus during periodic episodes of dental plaque mineralization. Other exogenous proteins transiently present in the oral cavity can also become incorporated during these mineralization events, including dietary proteins such as milk beta-lactoglobulin (101) and seed storage proteins. (102) Similarly, proteins within cooking vessels may become adventitiously preserved within calcified crusts (limescale) during evaporative comineralization (99) and during the corrosion of nearby items such as metal objects. (103,104)
In nearly all cases, mineralization is an important factor in protein long-term survival. Excluding exceptional cases from water-logged, (105,106) arid, (107,108) or very cold contexts, (109) proteins not encapsulated in a mineralized matrix generally do not persist over long periods, as has been shown experimentally for proteins applied to ceramics (110) and stone tools. (111−113) Beyond incorporation, it is also important to note that some proteomes are also altered during the incorporation process, whether by cross-linking during the tanning of leather, (114) heat denaturation during the cooking of foods, (115) or by autodegradation at the time of formation, as is the case for enamel. (116)

2.2. Processes of Decay and Diagenesis

Most organic material and proteins decay and are recycled into the environment when they are shed by a living organism or after an organism dies. (117) Degradation is primarily mediated by bacteria through enzymatic digestion, which occurs relatively quickly. (118) Experimental biodegradation of woolen fabrics and feathers has shown that even relatively robust proteins, such as keratins, which are hydrophobic and contain numerous disulfide linkages, do not survive for long under unfavorable microbial conditions. (119,120)
Only a very small percentage of the overall proteins are expected to persist in the archaeological record, (121) and those that do are generally mineralized, highly abundant, and/or have unusual properties. Type I collagen (COL1), for example, is the longest persisting bone protein, and it makes up >80% of the bone proteome (accounting for 20–30% of the mass of fresh bone), is heavily mineralized, and is arranged into a highly stable triple helix. Among dietary proteins identified in dental calculus, many are either protease inhibitors or belong to the seed storage superfamily, (102) both of which are known to be highly stable against proteolysis and thermal processing. Likewise, milk beta-lactoglobulin, which is perhaps the best attested ancient food protein, has a small molecular size and stability to changing pH levels and enzymatic degradation, (122) all properties that are known to contribute to protein survival under harsh conditions. (123) Following the initial stages of decomposition, surviving proteins are then subject to slower taphonomic processes that continue the diagenetic alterations. (124,125) In this way, nearly all ancient proteins undergo some degree of degradation or chemical damage.
Because of the diversity of proteins in terms of composition, chemical properties, size, shape, function, and incorporation (or lack thereof) into mineralized tissues, the taphonomic factors that drive post-mortem protein degradation and decay are highly variable and more poorly characterized than those for other ancient biomolecules, such as DNA. This “black box” of taphonomy is therefore an ongoing challenge for the analysis of ancient proteins, not because of a lack of research effort, but rather because of the immense complexity of the problem. Nevertheless, there are some factors that are known to play a consistent role in the protein degradation process: (1) local environment, including soil chemistry, pH, and water availability; (2) the chemical and structural composition of the matrix in which the proteins are incorporated; (3) the composition of the proteins individually and as a proteome; and (4) the local thermal history including time, temperature, and humidity. (126) These factors combine to create “diagenetiforms”, or diagenetically modified protein fragments, (127) formed through hydrolysis of peptide bonds and amino acid degradation, as well as racemization.
Protein fragmentation, the progressive, irreversible process of backbone cleavage into increasingly smaller pieces of the original protein, is among the most important forms of degradation. As the weakest covalent bond in a protein, the peptide bond is susceptible to spontaneous hydrolysis, with variable rates across the protein depending upon water accessibility to the peptide bond. This is mediated by the primary amino acid sequence, the protein secondary and tertiary structure, and surface stabilization by a mineral matrix. Protein preservation is generally better under conditions of limited water availability, such as in arid or frozen environments or where proteins are trapped in locally hydrophobic environments or in the intracrystalline fractions of biominerals. (9,126)
The other major form of protein degradation is the chemical alteration of an amino acid R group or the C or N terminus of a peptide. Such changes are myriad and incompletely characterized, (127,128) but the sheer diversity of potential chemical reactions can be appreciated by simply considering the enormous range of low molecular weight nitrogen-containing compounds formed by the diagenesis of starch storage tissues through so-called Maillard reactions. (129) Diagenesis therefore affects the chemistry─and more importantly the mass─of the affected amino acid, which can interfere with the recovery and identification of peptides using mass spectrometry. Rates of modification are again highly dependent on the primary amino acid sequence, secondary and tertiary protein structure, and surface stabilization of the protein or peptide. Indeed, it has been speculated that surface mediated preservation may promote the formation of novel condensed structures. (130) Additional modifications can be further introduced during the extraction process, either intentionally to chemically disrupt the conformation of the proteins (e.g., carbamidomethylation of cysteine by reduction and alkylation) or unintentionally through the production of undesired reactions (e.g., protein carbamylation by urea derivatives in the presence of heat).
Foundational studies on fossil invertebrates (131) and more recent studies of bone, enamel, dental calculus, and eggshell have provided insights into the range of diagenetic modifications present in ancient proteins, (9,13,92,121,132) with the most frequently identified being backbone cleavages and the deamidation of asparagine and glutamine. Other common diagenetic modifications are carboxymethylation of lysine (an advanced glycation end-product), conversion of serine to alanine, the conversion of histidine to hydroxyglutamate, the formation of N-terminus pyroglutamic acid, decomposition of arginine to ornithine, and various forms of oxidation, phosphorylation, dephosphorylation, hydroxylation, and dehydroxylation. (9,127,128) However, these represent only the forms of damage that are observable in mass spectrometry studies. Other forms of chemical modification that interfere with protein extraction and ionization are much less well understood and may mask pools of persisting but largely inaccessible proteins. (133) It is probable that many of the changes observed over time are also occurring in the kitchen, and therefore, it is worth paying attention to the expanding field of proteomics applied to food science. (134)

2.3. Methods of Recovery

In order to be detected and analyzed, proteins must first be extracted from the matrix to which they adhere or in which they are embedded. Numerous protein extraction methods are available, and their demonstrated success rates depend on the source and chemical properties of the proteins under experimental study. Compared to modern proteins, ancient protein extraction is further challenged by protein diagenetic alteration and the frequent incorporation of ancient proteins into mineral matrices. Protein loss is inevitable during this stage, both from an inability to fully “unstick” proteins from the matrix that aided their successful integration into the archaeological record and from differential recovery due to performance variation in extraction and digestion methods. Contamination may be introduced at this stage, and laboratory contaminants that have been previously observed include latex proteins from gloves, egg proteins from commercial cell lysis buffers, common laboratory reagents such as serum albumin, proteins from human sweat (e.g., dermcidin), and a wide range of keratins from human skin and sheep wool. Public lists of common laboratory contaminants, such as the common Repository of Adventitious Proteins (cRAP; https://www.thegpm.org/crap/), can aid in contaminant identification, but other potential sources of local laboratory contamination should also be considered. An awareness of potential contaminant sources and adherence to best laboratory practices is critical to mitigating laboratory contamination. (4,135)

2.3.1. Extraction Methods

Protocol development for ancient protein extraction is an active field with multiple methods in widespread use. When choosing an extraction method, sample type, size, and preservation should be considered, as well as the complexity of the proteome, the protein(s) of interest, and the amount of protein needed for analysis. The postdepositional history of the sample should also be taken into account, as well as potential chemical modifications introduced during the chosen extraction protocol. (136−141) In addition, because proteomic analyses often require less sample material than other methods, such as ancient DNA analysis, stable isotope analysis, and radiocarbon dating, protein extractions can often be performed on the leftover material or byproducts of these protocols. (139,142−145) Combining such protocols is desirable, as it reduces sampling demands on irreplaceable material.
For mineralized samples, such as enamel, bone, dental calculus, and shell, a demineralization step is generally required using either a weak acid or a chelating agent, such as ethylenediaminetetraacetic acid (EDTA). This is generally followed by protein solubilization using a variety of possible options, including heat, mechanical disruption, chaotropic agents (e.g., urea or guanidinium hydrochloride), detergents (e.g., sodium dodecyl sulfate, SDS), buffers, and salts. If the proteins are complex or are known to contain cysteines, reduction and alkylation steps are typically performed to irreversibly disrupt disulfide bonds. At this point, buffer exchange is frequently necessary to make the suspended or solubilized proteins compatible with downstream analysis, and different strategies for this are available including protocols based on the use of polyacrylamide gels, (95,146) filter-aided sample preparation (FASP), (91,102,147) gel-aided sample preparation (GASP), (148,149) single-pot solid-phase sample preparation (SP3), (150,151) or simply physical removal of the insoluble protein from the decalcification buffer in the case of collagen pseudomorphs. (152) This is then typically followed by enzymatic digestion of the proteins into peptides, followed by peptide purification, typically using C18 resin (commercially available as StageTips [Pierce] and ZipTips [Millipore]).
For nonmineralized samples, such as artist materials (binders, glues), mummified tissues, and parchment, simplifications to the protocol can be made. Demineralization steps can be avoided, and if protein solubilization is possible in a buffer compatible with mass spectrometry (e.g., ammonium bicarbonate or guanidinium hydrochloride), buffer exchange can be avoided, which mitigates protein loss. (137,146,153) Recently, bioactive films have been developed that allow “lab-on-plate” protein extraction directly from sample material such as artwork, which further simplifies extraction of surface-available proteins. (154)
In studies focusing on a small number of highly abundant proteins of interest, such as collagens in bone and parchment, additional simplifications can be made, even for mineralized samples. Less invasive techniques can be applied to sample loosely bound proteins (155−158) and even trace proteins left behind in storage bags and containers. (144,159) However, such techniques are more susceptible to ambient contamination, and because they target unbound and largely surface proteins, the recovered proteins are likely to be more degraded.
Prior treatments or chemical exposures that interfere with protein extraction and analysis can also be mitigated in many instances. For example, synthetic adhesives, which are sometimes applied by conservators to consolidate and stabilize fragile materials, can be removed with acetone prior to protein extraction. Likewise, nonproteinaceous chemical coextractants that interfere with mass spectrometry, such as soil humic acids, can be removed from both mineralized and nonmineralized samples using sodium hydroxide (NaOH) washes during early stages of the extraction process. (160−162)
Protein contamination can be a more challenging problem to address, and is especially troublesome for AAR studies (46,47) and PMF of eggshell. (87) For mineralized tissues, however, extraction methods can be modified to focus on only mineral-bound and encapsulated proteins. A strong oxidizing agent, such as sodium hypochlorite (NaOCl), can be applied to destroy proteins not encased within mineral, (9,163) leaving behind only intracrystalline proteins. (126) While not necessary in all cases, this aggressive decontamination approach can dramatically improve the proportion of endogenous proteins recovered, even as it reduces the total protein recovery.

2.3.2. Digestion and Digestion-Free Methods

To date, all mass spectrometry studies of ancient proteins have followed a “bottom-up” proteomics approach, meaning that the target of analysis is enzymatically digested peptides rather than intact proteins. (164) Most current protein mass spectrometers are best suited for analyzing peptides in the size range of 6–30 amino acids, and enzyme selection is based on maximizing peptide digests within this range. (165) Trypsin (which cuts C-terminal to arginine and lysine residues), alone or in combination with Lys-C (which reduces missed cleavages at lysine residues) are the most commonly used enzymes for general purpose protein mass spectrometry. Alternative enzymes are also available, (166) and enzymes such as collagenase, elastase, pepsin, chymotrypsin, Glu-C, Lys-N, and ProAnalase have been used in ancient studies to improve coverage of specific proteins or protein regions of interest. (76,167−169) However, proteins characterized by low complexity repetitive domains, as are common in mollusc shell, are difficult to sufficiently digest by enzymatic methods alone and may require additional chemical cleavage to generate peptides of suitable size for mass spectrometry. (170)
Digestion free methods can be used when the proteins are already broken down into fragment sizes suitable for analysis. This primarily occurs in cases of high diagenetic backbone fragmentation or when proteins are autodigested in vivo. A study of 3.8 Ma ostrich eggshell was the first to successfully apply a digestion free method to the recovery of highly degraded struthiocalcin-1 (SCA-1) and struthiocalcin-2 (SCA-2) proteins. (9) More recently, the method has been applied to enamel, including enamel from present-day periodontal patients. (171) Enamel is composed of >98% hydroxyapatite mineral, and a critical step in its maturation is the enzymatic breakdown of the proteins involved in its formation, such as amelogenin. (116) As such, its proteome is already in a fragmented and degraded state during life, with protein fragments in a size range suitable for mass spectrometry. The enamel proteome is also small, comprising only a few major proteins, making it more feasible to analyze and interpret than other proteomes when cleavage positions are variable or unknown. Consequently, digestion-free methods can be applied to enamel proteins and have been used to obtain high quality protein sequences from teeth spanning a wide time range. (13,14,94,172) Such studies of eggshell and enamel are providing our first glimpses of ancient proteins that have been minimally modified by laboratory methods.
True “top-down” proteomics, the measurement and interpretation of mass spectrometry data from intact and native proteins, has not yet been achieved for ancient samples. (127,173) However, ongoing advances in both technology and bioinformatics over the past decade are improving the feasibility of “top-down” approaches. (174)

2.4. Detection by Mass Spectrometry

Once digested peptides or protein fragments have been isolated and purified, they can be analyzed by mass spectrometry. Today, the two main workhorses of paleoproteomics are peptide mass fingerprinting by MALDI-TOF and shotgun proteomics by LC–MS/MS. With respect to cost, time, sensitivity, scale, and scope, each brings different strengths and weaknesses to the study of ancient proteins (Table 1).

2.4.1. MALDI-TOF and Peptide Mass Fingerprinting

Peptide mass fingerprinting (PMF) is a technique used to identify proteins by the masses of the peptides produced following enzymatic digestion. First developed in the 1990s, (175) PMF works best on individual proteins, where ambiguities in peak assignment are minimized, but it can also be applied to proteomes of reliable composition or with one or more dominant proteins, such as collagen in bone or keratins in wool and feathers. PMF was made possible by the development of the soft ionization method matrix-assisted laser desorption/ionization (MALDI) during the late 1980s. (176,177) MALDI represented a major breakthrough in protein chemistry, enabling large, nonvolatile molecules such as small proteins and peptides to be ionized without fragmentation for downstream mass spectrometry. Coupled with a time-of-flight (TOF) analyzer, the MALDI-TOF mass spectrometry system is a robust, simple, and sensitive instrument with a large mass range (175) that is ideally suited for PMF.
To measure protein digests for PMF, acidified peptides are spotted onto a MALDI plate together with a matrix, typically α-cyano-4-hydroxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB), that cocrystallizes with the peptides. The matrix is then excited with a laser causing the peptides to vaporize and ionize with a + 1 charge. Electromagnets then direct the ions into a time-of-flight tube, where they are measured by a detector. Their time-of-flight (which is related to their kinetic energy and mass) is then converted into a spectrum of mass-to-charge ratios (m/z) vs intensity, and the observed peaks are ready for analysis using a database of protein and contaminant sequences. (72,77,178)Figure 3a shows an example collagen PMF from archaeological animal bone. Informative peaks (markers) originating from the α1 and α2 chains of COL1 are highlighted. Using the principle of parsimony, the nine markers collectively can be used to allow a conclusive assignment to sheep (Ovis). Other (nonannotated) peaks visible in the spectrum include matrix peaks, nonmarker collagen peptides, and peptides from keratin contaminants, noncollagenous proteins, and autodigested trypsin. For a more detailed, step-by-step explanation of collagen PMF interpretation, see refs (7) and (77).

Figure 3

Figure 3. Example MS and MS/MS spectra obtained from archaeological samples. (A) Mass spectrum of sheep (Ovis) type I collagen obtained by MALDI-TOF MS from an archaeological small ruminant bone bone at the site of Tepe Yahya, Iran (YTC-248, Peabody Museum No. 986-7-60/22498). (B) Tandem mass spectrum of sheep (Ovis) β-lactoglobulin milk protein obtained by nano-HPLC–MS/MS from human dental calculus at the Iron Age pastoralist site of Marinskaya 5, Russia (MKA018). (C) Tandem mass spectrum of sesame seed (Sesamum) 11S globulin protein obtained by nano-HPLC–MS/MS from human dental calculus at the Late Bronze Age city of Meggido, Israel (MGD011). (102)

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Because PMF involves matching a pattern of peaks generated by tryptic peptides, and not sequence determination, it requires access to databases with good taxonomic representation, and identifications are made on the basis of parsimony rather than unique peptide matches. Due to the functional constraints of the protein, sequence variation of COL1 is low, purifying selection is high, and mutational saturation is a challenge for some clades. (179−181) COL1 thus carries only a weak phylogenetic signal, but if enough marker peptides are sufficiently preserved, taxonomic assignments are generally possible to the family level of birds, the family or subfamily level of mammals, and the genus or species level of fish. (7) For a review of the use of PMF in archaeology, see ref (7).
Despite its limitations, PMF approaches offer several important advantages over other methods. PMF requires very little sample material and is compatible with a number of minimally invasive methods, which will be described below. It does not require specialized facilities, and it utilizes an instrument that is currently widely available at many research institutions and university core facilities. It is also fast and inexpensive, which allows it to be performed at scale and with high throughput. This combination of features makes it a highly flexible method that can successfully support both small-scale budget-restricted projects on specific questions (182,183) as well as large-scale exploratory studies of thousands of samples. (184,185)

2.4.2. LC–MS/MS and Shotgun Proteomics

Tandem mass spectrometry (MS/MS or MSn), as applied in the context of paleoproteomics, is an approach whereby mass analysis is conducted at least twice while performing a dissociation process in order to characterize peptides in a protein mixture. The first mass scan (MS1) measures the m/z of the ionized peptides (called precursor ions) and selects some for fragmentation by dissociation and further measurement by a second mass scan (MS2) that determines the m/z of the peptide fragments (fragment ions). Depending on the method of fragmentation, different types of fragment ions are produced. Collision-induced dissociation (CID), which is among the most widely used fragmentation methods, yields mostly b- and y- fragment ions. The MS2 measurement of these fragment ions allow the peptide’s amino acid sequence to be inferred with the aid of a database (or even de novo under certain conditions (186)), which allows greater confidence in peptide identification than the m/z of the precursor ion alone. The MS2 spectra for two peptides recovered from ancient human dental calculus and analyzed by LC–MS/MS are provided in Figure 3. In both cases, a near complete y-ion series was observed, as well as a partial b-ion series, allowing the peptide sequences to be determined with high confidence. The first sequence is a highly specific match to the β-lactoglobulin milk protein in sheep (Figure 3b); the second sequence is consistent with the 11S globulin protein of sesame seeds (Figure 3c). The ability to accurately measure both precursor and fragment ions makes MS/MS a powerful technique for identifying ancient proteins.
Although tandem mass spectrometers had been available in various configurations since the late 1960s, it was not until the development of soft ionization methods such as MALDI and electrospray ionization (ESI) in the late 1980s that MS/MS could be applied to proteins. (187) Early uses of MS/MS in paleoproteomics utilized a variety of mass analyzers, including ion traps, quadrupoles, and TOFs (64,74,89) but had relatively low sensitivity and mass accuracy, which limited the number of proteins that could be identified. The commercial introduction of the Orbitrap mass analyzer in the mid-2000s and subsequent hybrid systems marked a major improvement in protein mass spectrometry (188) and dramatically improved the detection and identification of proteins in low biomass, complex mixtures, which are characteristic of ancient samples. Gains in ancient protein identifications were enormous from the first commercial model, the Thermo LTQ Orbitrap, which identified three collagen proteins in a mastodon bone in 2007, (189) to the subsequent Thermo LTQ-Orbitrap Velos, which enabled the identification of a proteome of more than 100 proteins in a mammoth bone in 2012, (91) and then the Thermo Q-Exactive Hybrid Quadrupole Orbitrap, which allowed the characterization of a metaproteome comprising hundreds of proteins in human dental calculus in 2014. (92) Current MS/MS systems used in paleoproteomics are even more powerful and typically consist of an ultrahigh performance liquid chromatography (UHPLC) system coupled to a nano-ESI that interfaces with a high performance (high resolution fast duty cycle) mass spectrometer. Greater chromatographic separation, fractionation of samples, and/or the use of ion mobility (190,191) can further enhance resolution, and alternative ionization methods, such as desorption electrospray ionization (DESI) and liquid extraction surface analysis (LESA), (192−195) offer additional capabilities, including ambient ionization and in situ analysis; however, these approaches have not yet been extensively explored in paleoproteomics.
Tandem mass spectrometry is well-suited for the analysis of ancient proteins from diverse sample types. It can identify high and low abundance proteins in complex mixtures, and it does not require reliable - or even known - protein composition prior to analysis. Because it involves the simultaneous analysis of many proteins, it can be used to achieve higher taxonomic resolution than PMF, which is particularly important for resolving vertebrate (14,21,196) phylogenies. Moreover, it can also be used to identify protein variants, PTMs, and diagenetic alterations. These features make it ideal for discovery proteomics applications, such as phylogenetic analysis of extinct hominids, (13,172) taxonomic identification of worked shell, (197) determination of unknown binders in artwork (137) and the identification of dietary proteins in pottery crusts (99) and dental calculus. (101,102) Beyond shotgun approaches, MS/MS can also be used to some extent to target ancient proteins of interest using multiple reaction monitoring (MRM; also known as selected reaction monitoring, SRM) (198−201) and parallel reaction monitoring (PRM). (202) Although current MS/MS approaches largely rely on data-dependent acquisition (DDA) for precursor ion selection, which maximizes the success of peptide sequence determination but limits the method’s reproducibility and quantitative potential, data-independent acquisition (DIA) approaches (203,204) are in now development for ancient protein analysis. DIA offers the potential to extend the dynamic range of MS/MS by generating data from more peptides, and especially lower abundance peptides, while also improving reproducibility and quantification. A DIA-based approach has recently been integrated into a new paleoproteomics workflow known as species by proteome investigation (SPIN), which enables rapid mammalian species assignment using LC–MS/MS. (205) Although currently limited in scope due to its computational complexity, further improvements in DIA development, improved databases, and the application of machine learning may soon allow DIA to become more mainstream in ancient protein studies.
The major downside of MS/MS in ancient proteomics is its significant infrastructure needs, time, and cost. Samples for MS/MS should be prepared in a dedicated ancient biomolecules laboratory, in part because the higher sensitivity of the instruments and the discovery nature of the research makes distinguishing ancient proteins from contamination more difficult. Highly specialized and expensive mass spectrometers are required that may not be widely available at local core facilities, and the instrument time per sample is high (an hour or more), limiting daily throughput. Currently, the costs of MS/MS are typically 30–50 times higher on a per sample basis than PMF, although new specialized applications, such as SPIN, are faster and more affordable. Despite its difficulties, the power and performance of LC–MS/MS, and most importantly its ability to provide sequence data, make it a highly valuable─and even indispensable─technique to answer many paleoproteomic questions.

2.5. Analysis and Interpretation of Data

With the exception of largely experimental work still in development, (186) nearly all mass spectrometry data analysis methods relevant for ancient proteins rely on the use of specialized software, protein sequence or peptide marker databases, and the selection of priors.

2.5.1. MALDI-TOF and ZooMS

Analysis of PMF data for the purpose of taxonomic identification is most frequently performed manually through the visualization of spectra using FlexAnalysis (Bruker Daltonics) or mMass (206) software. Unfortunately there is no centralized database or public repository of PMF spectra or markers at present, and consequently peptide markers (peaks that have been empirically demonstrated to be taxonomically informative) must be retrieved from literature searches. Taxonomic identifications are made by applying the principles of parsimony to the combination of markers observed. (77) Depending on the potential species present, additional tools have been recently proposed to help with identification based upon machine learning, hierarchical clustering, principal components analysis (PCA), and theoretical spectra matching. (207,208) During spectra interpretation, it is essential to consider the possibility of mixed or composite proteome representation (e.g., in the case of glues), and potential contamination must also be taken into account.

2.5.2. LC–MS/MS, Protein Identification, and De Novo Sequencing

The analysis of MS/MS data is usually conducted with the aid of software that matches precursor ion masses and individual MS2 spectra against a set of theoretical masses calculated from a protein database and preselected priors, producing a peptide match score, as well as other metrics. This approach requires the input of: (1) a database, (2) instrument parameters, and (3) search priors. Some software also integrates de novo sequencing, machine learning algorithms, or other alternative workflows, which can reduce the reliance on databases and enable the characterization of novel sequences. The most commonly used software for paleoproteomics are MASCOT, (209) MaxQuant, (210) SEQUEST, (211) PEAKS, (212) and Byonic. (213) Additional software, such as Scaffold, (214) can be used to further authenticate and filter the results by protein and peptide probability and by false discovery rate (FDR), and peptide identifications are typically manually validated by searching them against the NCBI nr database using BLASTp to ensure specificity.
Although reference proteins can be directly sequenced, the vast majority of protein sequence data available in major protein databases derives from genetic coding sequences (CDS) submitted to NCBI (GenBank), EMBL-EBI (EMBL-Bank), and DDJB, the three major public nucleic acid databases that together form the International Nucleotide Sequence Database Collaboration (INSDC). Other sources of genome-derived annotated protein sequences include NCBI RefSeq (215,216) and Ensembl Genomes, (217,218) as well as WormBase (219) and ParaSite (220) for parasitic nematodes, and VectorBase (221) for pathogen vector genomes. UniProtKB, the world’s largest public repository of protein information, aggregates data from the INSDC and releases it in two databases: (1) SwissProt, which contains manually annotated and reviewed sequences; and (2) TrEMBL, which consists of all remaining nonreviewed, automatically annotated sequences. Specific databases are also available for individual species proteomes (e.g., UniProt Proteomes, NCBI RefSeq), and custom databases can be created using curated lists of genomes, such as the Human Oral Microbiome Database (HOMD), (222) or metagenomes, such as NCBI env_nr. In addition, specialty databases for identifying common laboratory contaminants, such as the common Repository of Adventitious Proteins (cRAP; https://www.thegpm.org/crap/) and databases for assessing preservation in specific ancient sample types, such as dental calculus, (223) have also been developed. These databases contain different numbers of sequences, varying levels of metadata, and different database biases. (224) Decoy databases, or other integrated target-decoy search procedures, are included during analysis in order to calculate FDR. (225) Care should be taken to ensure that the selected databases are appropriate for the sample type. For example, studies of microbial substrates, such as dental calculus and paleofeces, or tissues that have been degraded by environmental bacteria, such as skeletal and mummified tissues, should include microbial proteomes in database searches in order to ensure that microbial proteins are not better matches for spectra putatively assigned to dietary or host-derived proteins. Likewise, investigations of pathogens (e.g., Mycobacterium tuberculosis) should also include protein sequences from related microbial taxa (e.g., soil Mycobacteria) in order to ensure taxonomic specificity. Discovery-based proteomics, especially when applied to complex metaproteomes, is particularly sensitive to database selection, and overly restrictive databases should be avoided in order to mitigate false positives.
Once one or more databases have been selected, instrument parameters and search priors are required. Instrument parameters correspond to the particular mass spectrometer used for the analysis, and include information about peptide ionization and MS1 and MS2 mass accuracies. Search priors relate to the sample itself, and consist of information about the enzymatic digestion, isotopic composition, and anticipated chemical modification of the peptides. This includes specifying the digestive enzyme (e.g., trypsin), which should match the experimental enzyme used, as well as the assumed fidelity, making allowances for missed cleavages (typically 1–3). The number of anticipated 13C atoms is typically preselected, and fixed and variable chemical modifications resulting in mass changes are also specified as priors. These include any intentional modifications introduced during extraction (e.g., carbamidomethylation of cysteine), common biological PTMs (such as oxidation and phosphorylation), and diagenetic modifications (such as deamidation and glycosylation).
While the ability to match against every known protein sequence and all possible chemical modifications would be ideal for ancient samples, computational effort scales linearly with the number of sequences in the database and exponentially when relaxing chemical modification and digestion parameters. Therefore, choices must be made to limit the search space to allow reasonable computational efforts. For example, in vivo and diagenetic backbone cleavage can be accounted for in searches by selecting enzyme options such as “semi-trypsin”, but this increases the search space of the algorithm. Likewise, searches can also be conducted in “error tolerant” mode, which allows for amino acid substitutions and unspecified chemical modifications, but this increases the FDR and can reduce the number of successfully identified ancient proteins. For ancient samples, error tolerant searches are generally reserved for assessing the range of chemical modifications in a sample prior to further analysis (e.g., refs (92) and (102)) or for determining novel sequences in already well characterized proteins (e.g., collagen (226)) or proteomes (e.g., enamel (13,172)).
In phylogenetic studies of taxa for which the exact protein sequence is unknown, software is available for de novo sequencing. (227) This is especially valuable for determining sequences in extinct species for which genomic data cannot be obtained (20,172,226,228) or authenticating PMF markers. (73,182,229) A variety of software has been used to perform de novo sequencing on ancient proteins, including PEAKS, Byonic, and MaxNovo. Algorithmic differences between the different software influence the accuracy of identification. The taxonomy of tryptic peptides identified by de novo sequencing can be inferred using tools such as UniPept. (230)
After a search is completed, potential identifications are evaluated on the basis of a number of metrics, including peptide match scores, the number of assigned peptides for a given protein, and the peptide and protein FDRs. Taxonomic assignment for high scoring identifications should be further validated for specificity using additional sequence alignment tools, a diverse database, and manual validation of critical spectra. It is essential to take into account potential database and search biases when interpreting results, especially when conducting discovery-based proteomics studies. Great care must be taken in selecting parameters to obtain optimal and accurate results, (231,232) and improbable results (233,234) should be subject to further scrutiny. Recommendations for minimum standards of authentication and validation are described in ref (4).

2.5.3. Taxonomic Discrimination

Once peptides are identified to proteins, their sequences can be used to infer taxonomy and to discriminate between related taxa. (20,73,86,88,235) The taxonomic resolution of a given set of peptide sequences can vary widely, however, depending on the evolutionary history of the protein and the specific evolutionary forces acting on it. Overall, protein sequences provide less taxonomic resolution than DNA sequences, but proteins can persist millions of years longer than DNA, they are biologically present in much higher amounts than DNA, and they are found even in acellular tissues (e.g., enamel, eggshell). As such, they are our most valuable form of molecular sequence data for providing successful and reliable taxonomic identifications in deep time fossils and in the study of processed and manufactured objects that have undergone activities that are destructive to nucleic acids (e.g., leather tanning, liming of parchment).
Overall, sequence change is generally more constrained and occurs more slowly in proteins than in DNA. This is because, unlike DNA, where noncoding regions and redundancy in the genetic code allow for mutations independent of selective pressures, amino acid changes directly affect the protein and─depending on the chemical properties of the altered amino acid and the location of the substitution within the peptide─can strongly influence the protein’s structure and function. (236,237) In addition, the synthesis or acquisition of specific amino acids for incorporation into proteins can have different metabolic costs depending on the organism and the environment. (238) This means that almost all proteins are under some level of selective pressure, and substitutions between amino acids with similar chemistry generally occur at a higher rate than amino acids with different chemistry. (239,240) Consequently, there is a higher probability for convergent, parallel, and back substitutions in proteins, which can result in distantly related species sharing the same substitutions (homoplasy)─a particular problem for proteins under high functional constraint. (241−244)
Protein sequence conservation among closely related taxa determines the level to which taxonomic discrimination is possible. In some cases, taxonomic discrimination is not possible because there are no sequence differences between a set of taxa for a given protein of interest. For example, wild (Ovis ammon, Ovis gmelini) and domesticated (Ovis aries) sheep have identical milk β-lactoglobulin protein sequences, and thus, taxonomy derived from this protein cannot be assigned at a level lower than that of genus (Ovis). Even in cases where sequence differences do occur, taxonomic discrimination may fall short of theoretical predictions because not all variant sites fall within peptides that are likely to be observed using mass spectrometry, either because the corresponding tryptic peptides are too long or too short or because they are too hydrophobic. For example, the COL1a2 amino acid sequences of domesticated horse and donkey differ by 4 out of 1038 residues in the mature protein. Theoretically, discrimination between the two species should be possible on the basis of these four residues, but in practice tryptic digests of this protein result in all four of these taxonomically informative amino acids falling in peptides that are unlikely to be detected by mass spectrometry. As a result, horses, donkeys, and mules (horse/donkey hybrids) cannot be distinguished using standard ZooMS techniques. In contrast, sheep and goat COL1a2 proteins, despite being even more similar (differing by only 2 out of 1038 residues), are generally distinguishable by ZooMS because their taxonomically informative residues fall on a tryptic peptide that is frequently observed by MALDI-TOF mass spectrometry. (73,78,245)
Taphonomic alterations and digestion efficiency can also further influence taxonomic specificity. Within the β-lactoglobulin protein, for example, the tryptic peptide TPEVD(D/N/K)EALEK is the most frequently recovered peptide containing a taxonomically variant site. (4) However, the residue that distinguishes cattle (D) from sheep (N) is unreliable in archaeological samples because a cattle aspartic acid (D) cannot be distinguished from a taphonomically deamidated asparagine (N). Thus, peptides bearing the aspartic acid residue must be provisionally assigned as cattle/sheep. Moreover, the lysine (K) residue in this peptide that distinguishes goats is also a tryptic cut site (which cuts the peptide into fragments too short for MS detection), and thus, a taxonomic assignment of goat can only be made if there is a missed tryptic cleavage in this peptide. Such complications must be factored into strategies for taxonomic discrimination of archaeological proteins, and as a result, the taxonomic resolution of ancient protein data in practice is often lower than would be predicted from protein sequence alignments alone.
In some cases, additional metadata, such as the time period or location from which the sample was obtained, can provide information allowing a greater degree of taxonomic specificity to be inferred. For example, Ovis β-lactoglobulin sequences obtained from locations outside the range of wild Ovis species, such as in colonial-era Americas, can be reasonably assumed to have originated from domesticated Ovis aries. Likewise, Holocene-era Bos sequences obtained from European Neolithic sites postdating the decline of aurochs (Bos primigenius) and outside the range of zebu (Bos indicus) and yaks (Bos mutus, Bos grunniens) can be reasonably assigned to domesticated cattle (Bos taurus). Such context-based inferences, however, must be applied with care, especially in places and periods where multiple species may have been present or where former species ranges are not well-known.
Finally, since mass spectrometry recovers peptides and not entire proteins, and these peptides have varying levels of taxonomic specificity, taxonomic assignments are typically made on the basis of parsimony, with the assumption that the peptides derive from the fewest number of species possible. In the case of endogenous tissues, such as bone, peptide sets for proteins such as collagen are assumed to derive from a single organism. However, the assumption that all peptides derive from a single organism does not hold in the case of manufactured or mixed proteomes, such as collagenous glues, dental calculus, or pottery food crusts. In these cases, taxonomic identification is undertaken by examining the full range of peptide sequences obtained from many proteins, and also taking into account biogeographical and archaeologically relevant prior information in order to make the most reasonable taxonomic identification(s) from the available data.

3. Applications in Paleoproteomics

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Although ancient proteins are studied in a wide variety of ways in the fields of archaeology, cultural heritage, and paleontology (for a review see ref (5)), most applications can be grouped into one of three broad categories based the nature and composition of the sample: (1) proteins, where a single protein is the primary target of analysis; (2) proteomes, where groups of endogenous proteins inherent to a tissue or substrate are studied (e.g., bone, enamel, shell); and (3) metaproteomes, where protein and proteome mixtures of diverse biological or manufactured origin are analyzed (e.g., dental calculus, paleofeces, pottery crusts, artist materials). Here, we describe each application in turn and highlight its uses, strengths, and challenges.

3.1. Proteins

A large proportion of ancient protein research has been dedicated to the detection and taxonomic assignment of highly abundant proteins that form the dominant structural components of tissues. PMF by MALDI-TOF is particularly suited to this situation, but when preservation is poor or when higher resolution sequence data is needed, more powerful tandem mass spectrometry approaches, such as MALDI-TOF/TOF or LC–MS/MS, can be applied. The most frequently analyzed ancient protein is COL1, a robust structural protein (246−248) that is capable of surviving more than 3 million years under ideal conditions. (10) Other proteins of interest include keratins (84) to identify wool, horn, hair, feathers, turtle shell, and baleen, and fibroin, (249) the dominant protein component of silk. In addition, the enamel protein amelogenin, which is important for tooth formation, can be used to determine the genetic sex of some mammal species. (94,250)
While mineralized tissues, such as bone, enamel, and shell, are frequently the target of ancient protein analysis due to their durability, some proteinaceous soft tissues are also suitable for analysis. Collagens, keratins, and fibronins are collectively the major components of textiles and parchments produced from primary and secondary animal products, such as hide, skins, leather, wool, fur, felt, and silk. These materials are often culturally important, but ephemeral and underrepresented in the archaeological record, (251) although their preservation can be enhanced by contact with antimicrobial metals such as copper. Paleoproteomic methods can increase their visibility by improving taxonomic identifications and identifying trace remains.

3.1.1. Collagens: Bone, Dentine, Antler, Ivory, Parchment, Leather, Gut, and Scales

ZooMS analysis of COL1 is the most frequently conducted type of paleoproteomic analysis, (178) and it can be conducted on almost any collagenous tissue, including mineralized tissues such as bone, dentine, antler, ivory, and horn core, (72,252,253) as well as nonmineralized tissues such as skin, parchment, leather, gut, scales, and other soft tissues. (156,160,254) It is especially useful for identifying material that has lost its diagnostic features, such as worked bone (159) and bone fragments, (185) and it can be used to screen large numbers of nondiagnostic fragments for species of interest. (184) It has also been proposed as one possible screening method for assessing collagen preservation prior to radiocarbon dating, (255) although ZooMS requires less collagen than either radiocarbon dating or stable isotope analysis. (139)
ZooMS identification using MALDI-TOF is performed using a database of taxonomically informative marker peaks, and it is important that the sequences of newly developed markers are first verified as authentic collagen sequences with taxonomically informative amino acid substitutions using MALDI-TOF/TOF or LC–MS/MS, (79,178) although there are cases where this has not been feasible. (182,256) Large mammals, particularly European species, make up a large proportion of the published markers, e.g., (72,257,258) but markers for other taxonomic groups are increasingly being developed, including for non-European large mammals, (79,252,259−261) rodents, (262,263) bats, (262) cetaceans, (80,264) marsupials, (265) birds, (77,82) fish, (81,182,256) amphibians, (266) and reptiles. (83,262,266,267)
Over the past decade, ZooMS has been used to answer a wide range of cultural heritage, archaeological, ecological, and paleontological questions. For example, ZooMS has been used to study the manufacture of worked bones, artifacts, and cultural heritage materials (159,245,252,254,259,260,268−274) and to better characterize archaeological faunal assemblages and past human–animal relationships. (80,82,185,257,263,275) It has been used to better define past domestic animal management strategies, (73,261,276−279) document the introduction of commensal species associated with human activities, (280−284) and identify the exploitation of wild species. (79,83,285,286) It has contributed to the reconstruction of past ecologies (182,256,262,265,267,287−289) and to the study of extinct megafauna. (196,265) ZooMS has also been notably used as a low-cost, high-throughput screening tool of bone fragments in large Pleistocene cave sequences, leading to the discovery of otherwise nondiagnostic hominid remains, (144,184,257,290) including the offspring of a Neanderthal mother and Denisovan father. (291,292) Finally, because ZooMS can be performed using minimally invasive sampling techniques, (154,156,157,159) it has proven a breakthrough technology in the emerging field of biocodicology, (293) the multidisciplinary analysis of parchment manuscripts, codices, and other historic documents. (156,208,294−303)

3.1.2. Keratins and Corneous β-Proteins: Wool, Hair, Feathers, Baleen, and Turtle Shell

α-Keratins and corneous β-proteins (CBPs, formerly β-keratins) are two of the most important structural protein classes in vertebrates after collagens, and they are the major components of hair/fur, nails/claws, horns, hooves, feathers, beaks, turtle shells, quills, and baleen. They are also present, along with collagen, in skin. (304−306) Like collagens, dozens of individual keratin and CBP proteins have been characterized. (307,308) They can also be taxonomically distinguished by PMF using MALDI-TOF. (309) Unlike collagen-producing cells, keratin-producing cells die after producing keratins and CBPs, and consequently keratinous tissues are not living and do not remodel. (304) Keratinous tissues do not mineralize, and even the hardest CBP tissues (e.g., turtle shells, beaks, claws, etc.) rarely contain minerals and therefore are more subject to degradation than mineralized proteins. (310) However, under favorable preservation conditions keratins and CBPs can preserve due their hydrophobicity and resistance to many proteases, (311) and they are also found archaeologically when embedded in mineral matrix produced by the degradation of nearby metal items (e.g., weapons, crowns, pins, buckles). (104,310)
MALDI-TOF marker peptides to identify furs and textiles were originally developed using PCA methods and have been subsequently verified using LC–MS/MS or MALDI-TOF/TOF, providing genus level resolution for some groups of mammals (104,109,251,309,312−317) and whale baleen. (318) The only taxonomic group with available CBP markers providing genus level resolution is sea turtles. (310,319) The use of immunological assays to detect wool on metal artifacts and from textile imprints in soils is currently being investigated. (320,321) Increasing understanding of keratin and CBP diversity, for example differences in sheep wool pigmentation and curl/crimp related to domestication, selective breeding, and diet (322,323) and keratin texture variation associated with body location, disease, and age in humans, (324,325) may allow more information to be gleaned than just taxonomic classification.

3.1.3. Fibroin: Silk

Silks are structural proteins, composed of highly repetitive β-sheet motifs interspersed with flexible domains. Raw silkworm silk is composed primarily of two proteins, fibroin and sericin, with several additional proteinases and functional proteins making up a minor component. During textile production, the nonfibroin proteins are removed, leaving fibroin as the dominant protein in archaeological silk. (251,326) While silks in modern and historic periods all derive from a single domesticated silkworm species from China (Bombyx mori), a number of other insects can also produce silk, including other wild silkworms and moths (especially Bombyx mandarina, Samia cynthia, Antheraea sp., and Philosamia sp.) and spiders (e.g., Nephila clavipes and Araneus diadematus), and use of these silks has been documented historically and archaeologically in Asia, India, Europe, North America, and Australia. (251,327−330) Silks from B. mori and Antheraea pernyi can be distinguished proteomically by LC–MS/MS and immunological assays. (251,331) This has allowed silk textiles and their species of origin to be identified from sediments with textile imprints (249,332) as well as trace amounts of textile in contact with metal artifacts. (333,334) Further work on the characterization of fibronins from other species has the potential to greatly improve the understanding of silk production and trade and silkworm domestication.

3.1.4. Amelogenin: Sex Typing of Humans and Other Mammals

Amelogenin (gene AMEL) plays an important role in enamel formation and mineralization in newly secreted enamel. During enamel maturation, amelogenin is cleaved by proteases and trapped within the enamel matrix. (116,335) In monotremes, marsupials, and nonmammalian species, AMEL is an autosomal gene, while in eutherian mammals it is located on the sex chromosomes. (250,336) In most mammals, only AMELX is functional, and AMELY expression is low. In some species, there are no sequence differences between AMELX and AMELY. Other species have lost AMELY entirely. However, for species with sequence differences between AMELX and AMELY and where AMELY is expressed, protein analysis can allow sex descrimination. (336,337) These species include humans, (338,339) cattle, (340) bison, (341) sheep, (342) goats, (343) deer, (342) pigs, (344) horses, (345) and bears. (346)
Before the widespread application of LC–MS/MS to archaeological samples, attempts to use MALDI-TOF/TOF to identify both AMELX and AMELY for sex determination were met with minimal success. (347,348) Subsequent efforts using LC–MS/MS have proven much more productive for sex determination in ancient humans (94,171,198,202,349−354) and archaic hominids. (172) Sex determination for other species has been limited to date, but includes extinct species of rhinoceros (Stephanorhinus sp. (14)), probocideans (Notiomastodon platensis), and rodents (Myocastor coypus (355)). Although there are concerns that the method could produce inaccurate results due to the presence of low frequency of AMELY deletion variants in some populations, (356,357) sexing using amelogenin is generally a robust technique, and it is an important biomolecular tool in cases where mophological sex determination is not possible, especially for incomplete skeletons and juveniles, (94) or when ancient DNA analysis is unsuccessful or infeasible. (354)

3.2. Proteomes

The first ancient proteome, from mammoth bone, was published in 2012. (91) Consisting of more than 100 proteins, it was a singular achievement and marked the field’s transition from the study of ancient proteins to true paleoproteomics. Proteomes are the suite of proteins present in a given tissue, and while the genome of an organism remains relatively constant throughout the body and lifetime, the proteome can differ substantially. Proteomes consist of protein mixtures, which often have a high degree of complexity and a wide dynamic range of protein expression. (358,359) LC–MS/MS approaches are necessary to profile proteomes, but comprehensive proteome characterization is still highly challenging even for modern proteomes, let alone ancient ones. Nevertheless, significant progress has been made, particularly toward characterizing the proteomes of ancient bone, enamel, eggshell, and mollusc shell, with mummified tissues being less explored. Although proteome data can be used to provide taxonomic assignments, they are also capable of addressing much more complex questions regarding phylogenetic relationships, health, and aging and development. Below, we describe the current state of research on ancient proteomes and explore their applications.

3.2.1. Bone and Dentine

Bone and dentine have similar developmental origins and correspondingly similar, but not identical, proteomes. (141,360) While they are both dominated by the structural protein COL1, they also contain a diverse array of hundreds of other collagens and non collagenous proteins (NCPs). (100,361−363) Many proteins are shared between bone and dentine, including interstitial fibrillar collagens (e.g., types I, III, and V), as well as proteins that support collagen fibril organization (e.g., lumican, LUM) and facilitate biomineralization (e.g., biglycan, BGN; fetuin-A, AHSG). Other proteins are more tissue specific, such as periostin (POSTN), which is disproportionately expressed in bone periosteum, chondroadherin (CHAD), a cartilage-associated protein expressed on bone articular surfaces, and asporin (ASPN), a protein that facilitates tooth attachment to the periodontal ligament. Plasma proteins and blood clotting proteins are also typically present in bone and dentine proteomes, including prothrombin (F2), coagulation factor IX (F9), and coagulation factor X (F10). To date, ancient bone proteomes have been studied in humans, (96,168,258,364,365) mammoths, (91,366) moas, (128) cattle, (121,132) horses, (12,168) turkeys, (367) rabbits, (367) squirrels, (367) extinct rhinoceros, (14) and ancient dentine proteomes have been studied in humans (92,141,365) and extinct rhinoceros. (14)
Because many of the NCPs and some of the collagens have higher mutation rates than COL1, they are better targets for phylogenetic reconstructions, especially between closely related species. (258,368) Analyses of bone and dentine proteomes have successfully aided in resolving the phylogenetic relationships of extinct megafauna, (14,20,21,196) including archaic hominins. (258,365) In addition, bone proteome-scale analyses using new high-throughput LC–MS/MS workflows, such as SPIN, show great promise for enabling genus and species-level taxonomic assignments of nondiagnostic bone. (205)
Proteome-level analyses can also be used to detect altered bone proteomes, such as those that occur in association with changes in activity levels, (369) health and disease, (370) and age. (371) Chronic bone infections, such as osteomyelitis, and bone cancers are known to alter the bone proteome, (372−375) and a number of studies have attempted to diagnose archaeological cases of cancers based on proteomic evidence, (96,376) although the results so far have been mostly qualitative and nonspecific. Changes in bone proteome composition and chemical modifications also occur with advancing biological age across multiple tissues, including bone marrow, (371,377) and composition changes and modifications correlated with biological age and post-mortem interval have been observed in the proteomes from modern and archaeological skeletal material. (141,258,364,378−382) However, bone is one of the poorer characterized tissues in the Human Proteome Organization (HUPO) Human Proteome Project (383) and proteome changes observed in archaeological bone samples could be the result of diagenetic factors in addition to biological age, physiological stress, life history traits, and disease. More work is needed to assess the reliability of chemical modifications and proteome composition as signals in archaeological samples.

3.2.2. Enamel

Enamel is the hardest tissue in the vertebrate body. (384) Composed mostly of hydroxyapatite crystals, the amount of organic matrix in mature enamel is very small, making up less than 2% of its total mass. (385) Enamel has a small proteome, consisting of only five abundant proteins: amelogenins (AMELX and AMELY), ameloblastin (AMBN), amelotin (AMTN), enamelin (ENAM), and odontogeneic ameloblast-associated protein (ODAM). Of these, amelogenin is the most abundant and best characterized, comprising 90% of all enamel protein. (385) Other proteins, including keratins (e.g., KRT75), (386,387) as well as collagens and blood proteins, (116) have also been detected in enamel at trace levels, but the latter likely represent contamination from dentine. Enamel proteins are degraded in vivo by the matrix proteases enamelysin (MMP20) and kallikrein related peptidase 4 (KLK4), resulting in only enzymatically cleaved proteins being present in mature enamel. (384,385) These degraded proteins are concentrated in the enamel tufts at the dentine–enamel junction (DEJ) and along hydroxyapatite rod sheaths. (386) Although early attempts to directly analyze enamel proteins from modern and archaeological teeth typically only detected amelogenin, (348,388) methodological improvements have increasingly allowed the recovery of a richer enamel proteome. (14,94,171)
Due to the protective nature of enamel in comparison to bone, enamel proteins are some of the longest surviving proteins in the vertebrate body. (171) Although the enamel proteome is small and carries less phylogenetic resolution than other tissues, such as bone, ancient enamel proteins have nevertheless been used to successfully determine the phylogenetic relationships of extinct species, (13,14,172,355) and enamel proteins are especially valuable in cases when DNA or other proteins are unlikely to survive. (389)

3.2.3. Avian Eggshells

Like enamel, the shell biomineral matrix provides the potential for better protection from water and thus degradation of proteins than bone. (9) As such, eggshell holds great promise for surviving into deep time, and eggshell currently holds the record for the oldest successfully determined and independently verified peptide sequence─from a 3.8 Ma ostrich eggshell in East Africa. (9)
Avian eggshells mineralize around the egg membrane in a multistage process mediated by the organic matrix proteins. (390,391) Although ∼500–1000 proteins have been identified using proteomic methods in fresh eggshell, (392,393) considerably fewer are typically identified in archaeological samples. (86) Prior to analysis, archaeological eggshells are typically treated using bleach to remove intercrystalline components, leaving only intracrystalline proteins for analysis. LC–MS/MS analysis of eggshell has recovered a wide range of eggshell proteins, notably ovocleidins, ovocalyxins, ovalbumin, struthiocalcin, rheacalcin, ansocalcin, ovomucoid, ovotransferrin, ovostatin, and mucins, among others. (86)
Further characterization of eggshell proteomes using both MALDI-TOF and LC–MS/MS (86,87) have allowed the development of a large number of marker peptides that can reliably distinguish common bird taxa (most importantly chicken, duck and goose) using PMF. (394−397) PMF is a rapid and inexpensive method for identifying archaeological eggshell, but due to the complexity of the eggshell proteome and the limited number of characterized species, not all eggshell can be identified using this method. When MALDI-TOF analysis is unable to taxonomically identify the species, LC–MS/MS analysis can be conducted on those samples that have distinct MALDI-TOF spectra in order to facilitate identification. (398,399) Success will increase as more bird genomes are sequenced and deposited in genomic and proteomic databases. (392)
Eggshell proteomes are powerful means to increase the taxonomic resolution of archaeological eggshells and allow more nuanced interpretation of the interactions between humans and birds at archaeological sites. For example, because diet and age influences eggshell strength and quality, (400) future studies of archaeological eggshell may be able to provide insights regarding the early husbandry and feeding practices of domesticated birds, such as turkeys, (401) as well as captive wild birds subjected to intensive breeding programs, such as scarlet macaws. (402,403)

3.2.4. Mollusc Shells

Molluscs have evolved a biomineralized exoskeleton, or shell, that provides support, protection, and defense and also serves as a reserve of calcium ions. (404) Important to proper shell formation are the shell matrix proteins that can persist in the intracrystalline matrix of biomineralized shells, where they are protected from contamination and degradation into deep time. (405−409) Shell matrix protein sequences are diverse and therefore have the potential for providing genus or even species-level taxonomic resolution. (404,410,411) Recent protocols have been developed that can successfully extract shell matrix proteins (“shellomes”) from worked and unworked shells for analysis by MALDI-TOF and LC–MS/MS. (88,197,412) Although identification is currently limited by the lack of available reference data, (411) “palaeoshellomic” analysis holds great potential for revealing humans’ longstanding relationship with shell and shellworking, (197) which stretches back more than 100,000 years, (413) and will aid in the better understanding and resolution of amino acid geochronology. (126)

3.2.5. Mummified Remains

Although mummification is relatively uncommon, it does occur under a variety of artificial and natural conditions, providing a rare opportunity to study ancient soft tissues. (414) The most frequently preserved and analyzed soft tissue is skin. Although most proteomic studies of skin (and treated skin products, such as leather and parchment) have focused only on collagens, skin is a complex tissue with a diverse proteome that also includes a large number of less abundant proteins. (415,416)
Using LC–MS/MS, much of the ancient skin proteome can be accessed, and studies of human skin to date have focused on characterizing proteome preservation in artificially (417) and naturally (418) mummified skin, with an emphasis on documenting proteins associated with innate immunity. Studies of animal skins have gone beyond taxonomic assignment to further identify fetal proteins differentially expressed in the months just before and after birth (e.g., RPN2, HBBF, HSP90A), allowing the identification of calf skin and an estimation of the age of death of the animal. (106,156) However, despite the large number of questions that can be potentially addressed by analyzing the proteomes of ancient skins, hides, and parchments, the study of ancient skins has continued to focus mainly on collagen. Studies of ancient skins are challenged by the unavoidable ubiquity of modern human skin and hair proteins, and the interpretation of proteins from ancient skins requires a good understanding of their excavation and curation history in order to account for contamination. Additionally, until very recently, sampling of ancient skins required destructive techniques that were strongly discouraged by museums and archives. Recent successes with minimally invasive EVA films on mummified tissues (419) and PVC rubbings on parchments (156) are changing the landscape of ancient skin studies and will likely increase the number of available samples that can be analyzed. (419)
In addition to skin, other mummified soft tissues and organs have also been documented and occasionally analyzed using proteomic techniques, including muscle (417) and stomach tissue. (420) More surprising are the hundreds of preserved brains that have been characterized, (421) with likely thousands more being preserved around the world. (422) At the time of writing only three have undergone proteomic analysis, (422−425) with highly variable numbers of proteins recovered. The success of brain preservation is likely due to the formation of protein aggregates that provide protection against degradation. (422,424) While soft tissues still remain relatively rare in comparison to other sample types, protein markers for health, age, and life history could potentially be developed.

3.2.6. Plant Macroremains

Plant macroremains (e.g., seeds, fruits, wood) can preserve under extraordinary conditions, such as in waterlogged or charred states, or in cases of extreme aridity or cold. (426) Although modern seeds contain proteins numbering in the hundreds to low thousands, (427,428) protein recovery reported from ancient seeds has been much lower. For example, only six plant proteins, consisting of 2S albumin, 7S and 11S globulins, peptidase A1, and a nonspecific lipid transfer protein, were identified in waterlogged grape seeds from medieval York, England, and none from Byzantine Lecce, Italy. (429) Overall, protein preservation of ancient seeds has been found to be relatively low compared to other biomolecular classes, such as carbohydrates and lipids, (429−431) but improved methods of protein recovery and recent successes in identifying plant proteins from residues (99) and stains (432) warrant renewed efforts to analyze ancient plant proteomes from plant macroremains.

3.3. Metaproteomes

Metaproteomes are protein mixtures deriving from more than one proteome. The vast majority of metaproteome research focuses on the study of mixed microbial communities, such as those found in host-associated and environmental microbiomes, (433) but many of the same methodologies also apply to mixed proteomes of made or manufactured origin, such as food crusts and artist materials, and so these materials will also be considered here. Also considered here are proteins recovered from microbially infected and diseased tissues. Studies of metaproteomes are among the most exciting and fastest-growing applications in paleoproteomics, but they also pose unique challenges for protein identification and authentication.

3.3.1. Microbiomes

Microbiomes are diverse microbial consortia that form stable complex communities. They may be host-associated, such as the oral and gut microbiomes, or environmental, such as the soil microbiome, and they can vary spatially within a given site or across different conditions. For example, within the oral cavity, the microbiota present on the gums differs from that on the tongue which further differs from that within dental plaque, and even within dental plaque, there are differences between supragingival and subgingival dental plaque. (434,435) Likewise, soil microbiota differ greatly across different environmental conditions, such as deserts, forests, and agricultural fields. (436) In spite of, or perhaps because of, this enormous capacity for diversity and variation, the study of modern and ancient microbiomes is highly valuable. (437)
The first metaproteome of an ancient microbiome was characterized from human dental calculus, (92) a form of calcified dental plaque that forms naturally during life. (438) Numerous microbial proteins were identified, including virulence factors specific to periodontal pathogens, such as TfsA and TfsB from Tannerella forsythia, which were independently confirmed using metagenomic techniques. However, despite searching the metaproteomic data against all of UniProt, it was clear that oral microbial proteins were being underidentified due to insufficient representation of oral bacteria in the database. The creation of a custom database based on the translation of genomes in the HOMD (222) produced improved microbial results, and another custom database created by translating metagenomic data from the same samples yielded even more microbial identifications, but the latter approach failed to yield a better understanding of the metaproteome since most of the translated sequences lacked annotation or were categorized as “hypothetical” proteins. Subsequent investigations of dental calculus have confirmed that it preserves an extremely rich and diverse microbial metaproteome, (439,440) but underdeveloped databases (both proteomic and genomic) limit the number of identifications and interpretations that can be made at present.
Beyond microbial proteins, dental calculus also preserves a rich mixed proteome of saliva and gingival crevicular fluid, including proteins involved in host immunological response to dental plaque and tissue destruction due to periodontal disease. (92) Many of the host proteins identified in both modern and ancient dental calculus are expressed by neutrophils, (92) the major cell type involved in periodontal innate immunity. (441,442) Nonimmunity related host proteins in dental calculus include α-amylase, a starch digestive enzyme expressed in saliva. (92)
In addition to microbial and host proteins, dental calculus also variably contains dietary proteins. (149) The first dietary protein to be identified in ancient dental calculus was beta-lactoglobulin, a protein highly specific to milk. (235) Milk proteins were subsequently identified in archaeological dental calculus from Europe, (149,440,443,444) Africa, (223) and Asia, (101,445) contributing to an understanding of how dairying arose and spread in prehistory. Plant proteins have also been identified in dental calculus, and the number and diversity of identified plant proteins have steadily grown as both methods and instrumentation have improved. The first study to recover plant proteins from dental calculus surveyed the Iron Age to medieval periods in Britain and identified dietary proteins from oats, peas, and cruciferous vegetables only in the youngest samples. (149) Subsequent studies in the Bronze and Iron Age Levant identified not only dietary staples, such as wheat and sesame, but also spices, oilseed, and fruit likely introduced through long-distance trade. (102) Other dietary proteins that have been identified to date in archaeological dental calculus include chicken egg ovalbumin (444) and ruminant hemoglobin. (149)
Although the majority of ancient microbiome research has focused on dental calculus, paleofeces are also now being explored. A recent study of Alaskan dog paleofeces, for example, identified a wide range of host proteins, including proteinases, peptidases, and lipases related to gastrointestinal digestion, as well as dietary proteins deriving from Salmonidae fish that indicate the consumption of fish muscle, guts, and eggs. (446) Few bacterial proteins were identified, as these were intentionally depleted prior to analysis. Cytochemical staining indicates that proteins are present throughout paleofeces, (447) and prior work on paleofeces using immunoassays has suggested that gastrointestinal parasites are also accessible through proteomic techniques. (448−451) Future analyses of paleofeces may yield insights into the structure and function of the ancient gut microbiome, as well as reveal richer information about health and diet. (452,453)

3.3.2. Residues, Crusts, and Food Remains

Ceramic cooking vessels have been shown to preserve dietary lipids and small metabolites (e.g., miliacin, tartaric acid) over long periods, allowing the tracking of prehistoric food practices, such as fish processing, (454−456) the spread of millet, (457) dairying, (458−460) vegetal oil storage, (461) and wine production. (462−464) However, efforts to recover dietary proteins from similar vessels have been met with only limited success to date. (465−467) It appears that proteins generally do not persist in, or cannot be extracted from, pottery. (133,468)
In contrast to the ceramic fabric itself, food crusts provide a much more promising target for molecular analysis, (456,469) and calcified food crusts appear particularly suitable for proteomic analysis. A recent proteomic study of 8,000-year-old calcified deposits (limescale) built up on the interior surfaces of cooking vessels at the Anatolian Neolithic site of Çatalhöyük yielded a remarkably diverse range of food proteins, (99) including milk whey (e.g., β-lactoglobulin, α-lactalbumin), curd (e.g., α, β, and κ caseins), and fat globule-associated proteins (butyrophilin subfamily 1 member A1), as well as meat/blood proteins (hemoglobin) and a wide variety of plant proteins (e.g., hordeins, legumins, serpin-Z4) from cereals and legumes. Dietary proteins were found to be concentrated within the calcified crusts on the interiors of the vessels, and the analysis of noncalcified ceramic fabric from the same vessels produced comparatively few proteins. Importantly, this study showed that ancient cooking vessels were used and reused to cook a wide range of plant and animal foods, with few vessels suggesting specialized use. Although lipid analyses, especially those based on isotopic analysis of C16:0 and C18:0 fatty acids, may be confounded by such culinary practices, proteomics is ideally suited for sorting out and distinguishing these food mixtures. (99)
Beyond food crusts, foods have occasionally survived relatively intact under exceptional environmental conditions, such as those found in the Taklamakan desert in western China. There, whole pieces of kefir-like cheese were found adorning mummies associated with the Bronze Age Xiaohe horizon, (107) and dried milk was identified lining the interiors of grass baskets. (108) In addition, sourdough bread made of barley and millet was preserved at the Subeixi cemetery during the subsequent Iron Age period. (95) LC–MS/MS analyses of these remarkably preserved foods yielded proteins not only integral to the food itself but also provided insights into the fermentation of these foods by lactic acid bacteria and yeasts, providing an unprecedented glimpse at prehistoric culinary technologies. Other examples of ancient foods directly analyzed by proteomics include gut content studies of unusually preserved corpses, such the Tyrolean Iceman Ötzi, an Alpine glacier mummy dating to the Chalcolithic, (420) and Tollund Man, an exceptionally well preserved bog body from the Danish Early Iron Age. (470)

3.3.3. Infections and Diseased Tissues

Ancient DNA has contributed to major advances in paleomicrobiology and pathogen genomics, (437,471) and has led to the molecular identification and characterization of more than a dozen infectious pathogens, including Yersinia pestis, Mycobacterium tuberculosis, Mycobacterium leprae, Helicobactor pylori, Treponema pallidum, Salmonella enterica, Plasmodium falciparum, hepatitis B virus (HBV), and variola virus. (472) By comparison, paleoproteomic studies of ancient pathogens are in their infancy, but offer the potential to study disease pathophysiology and clinical presentation.
Most proteomics-based studies to date have focused on infectious diseases that produce visible pathologies in the skeleton, particularly tuberculosis. However, initial enthusiasm for the identification of M. tuberculosis proteins by PMF (473,474) was later tempered by LC–MS/MS studies that showed a lack of peak specificity and the difficulty of distinguishing M. tuberculosis peptides from those of other soil mycobacteria. (475) Other studies tried to achieve higher specificity by applying an immunoassay approach, but lacked controls for environmental mycobacteria. (476) Recently, promising results were obtained for M. leprae infection using a combination of paleogenomic and paleoproteomic techniques applied to dental calculus. (477) In this study of a middle-aged adult female with osteological indications of leprosy, a 6.6-fold coverage M. leprae genome was reconstructed using ancient DNA techniques, and LC–MS/MS analysis recovered evidence of four mycobacterial proteins. Although none of the mycobacterial peptides were specific to M. leprae, taken together the osteological, genomic, and proteomic data provide a convincing portrait of a leprosy infection. Beyond its findings, the study highlights the difficulties of studying infectious diseases using a proteomics approach. Just as the field of ancient DNA only overcame its challenges in paleopathology with the transition to whole genome sequencing, (478) the field of paleoproteomics will also need to achieve greater pathogen proteome coverage before its full potential can be reached.
Beyond pathogen proteins, there has been recent interest in characterizing innate immune proteins within mummified and skeletal remains as a proxy for inflammation and disease. (364,418) While promising, more work is needed to understand the natural levels of immune proteins within healthy tissue, both from modern and ancient contexts, before such findings can be fully interpreted.

3.3.4. Cultural Heritage Materials and Works of Art

The application of mass spectrometry and other methods for inferring proteinaceous components of artwork and other cultural heritage objects has been comprehensively reviewed elsewhere. (479−483) Both MALDI-TOF and LC–MS/MS can be used to analyze artwork and cultural heritage materials, and their uses are described briefly below.
In ancient and historic times, proteinaceous materials (e.g., milk, eggs, blood, and gelatin from skin or bone) were widely used as binders for pigments in works of art and for building materials, such as mortars. Understanding the composition of these materials allows insight into past practices and provides information that will inform curation choices. Proteomic analysis of paintings has allowed identification of many widely used binder proteins, including caseins and beta lactoglobulin from milk, collagens from gelatin, vitellogenins, apolipoproteins, and low-density lipoprotein receptor from egg yolk, and ovalbumin, ovotransferrin, and lysozyme from egg white. While some studies have found that binders from a single source were used, (89,98,157,484−488) many binders are composed of mixed sources, such as milk from at least two different species or various combinations of milk, eggs, and gelatin. (97,199,489−497) Proteins introduced unintentionally during the creation of binders, such as muscle or blood proteins in collagen-based glues, might be able to provide insight into gelatin production or the addition of other animal products into the binders. (489) Beyond paints, binder proteins were also added to construction materials, such as mortars. Blood and milk are frequently recorded as mortar additives, and they have been detected in ancient mortars using a variety of techniques (498−500)
There are many historically recorded recipes for binders, and proteomics can illuminate the use of different recipes as they correlate with pigment color, type of canvas or statue, properties and availability of the binder, and cultural and personal choice. (97) Currently this is limited to some extent by detection bias and the limited number of samples analyzed. Because caseins and some egg proteins are not detected well with trypsin alone, multienzyme digests using both trypsin and chymotrypsin can improve detection of these binding materials. (169) In cases where the proportion or preservation of binder proteins is very low, targeting techniques such as MRM can help improve the detection of binder peptides, and this has proven particularly useful for identifying egg proteins in cases where their presence was suspected but not previously detected. (199) The recent development of low cost, minimally destructive sampling techniques that can be conducted without specialist training (154,157,169,490,501−503) promises to make proteomic studies of artwork more feasible, and further development of PMF using MALDI-TOF will facilitate larger scale data collection.
In addition to paint and mortar binders, a number of other historical and archaeological cultural heritage items have also been analyzed using proteomics techniques. Collagenous glues made from both mammal and fish gelatin have been identified in historical and archaeological samples. (201,504,505) Many other varying items have been analyzed including cosmetic sticks, (506) metal coated gut threads, (507) organic coatings on skulls and artifacts, (508) a birth girdle, (432) photographs, (503) and collections of museum items. (254) Proteomic analysis of these items can assist in better understanding the knowledge, processes, and choices used to create material culture.
Proteomics is also proving useful for understanding past conservation practices and guiding future efforts. For example, a recent study of historically conserved calfskin revealed cross-linked calf-rabbit collagen peptides, indicating that formaldehyde and rabbit glue had been used to conserve the piece. (509) In addition, identification of mixed and even mislabeled glues as part of conservation practices is just beginning to be explored. (490) Proteomic techniques can also help identify fungi and bacteria living on the surface of cultural heritage objects, such as parchment, (510) and in future it may be possible to use this information to identify objects at risk and to enhance methods of conservation.

4. Current Challenges

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Paleoproteomics is revolutionizing the way we study the past and providing unprecedented insights into evolution, phylogeny, human economies, cuisine, art, and other forms of material culture. However, current inefficiencies in protein recovery and measurement, limits of instrumentation, and insufficient databases and computational power hamper our ability to fully access ancient proteomes. Combined, these problems can result in the failure to identify key proteins that are present in a sample, create biases of detection, or limit the ability to quantitatively analyze archaeological and historical proteomes. Here we discuss major challenges still to be overcome with respect to the detection, identification, and authentication of ancient peptides and proteins.

4.1. Protein Detection

In order for proteins to be detected they must become incorporated into the archaeological record, survive over time, denature, solubilize, and be digested during the extraction process, ionize in the mass spectrometer, and fall within the instrument’s dynamic range of detection. These are hard limits for mass spectrometry, and any peptides that remain undetected are unrecoverable. Biological and diagenetic variation can lead to proteins and peptides being unequally incorporated, degraded, extracted, digested, and ionized in ways that are influenced by the protein’s amino acid sequence, hydrophobicity, protein structure, chemical modifications, interactions with a mineral matrix, and choice of extraction method. (9,140,366,511,512) Instrument design and performance also limits the detection and reproducibility of findings. (92,188,191,513,514) Some of these factors can be anticipated, while others not.
Biases related to protein size and sequence are among the most predictable. For example, some proteins (e.g., COL1) are extremely large, and thus produce many peptides, making their detection more likely than smaller proteins, even if both proteins are present in equal quantities (Figure 4A). Other proteins, such keratins, have a large number of cysteine disulfide linkages that can be difficult to fully break during extraction, resulting in underdetection (Figure 4A). Following digestion, some peptides can be predicted to have low recoverability because they are either too small or too large for efficient ionization and measurement by a given instrument (Figure 4A). In addition, diagenetic changes can also alter protease cut sites, changing detectability. For example, the positively charged side chains of Arg and Lys are prone to deamination and glycation, which contributes to missed cleavages and reduces the efficiency of trypsin in older samples. In vivo autodigestion (e.g., enamel proteins) and diagenetic backbone cleavage also alter expected peptide profiles.

Figure 4

Figure 4. Sources of bias in ancient protein identification. Differences in the density of enzymatic cut sites, number of disulfide bonds, and protein length influence protein detectability (A). The representation of proteins across taxa is highly uneven in major databases, such as UniProtKB (B,C). (A) Comparison of four Ovis aries (sheep) proteins of archaeological interest: AMELX, used for determining sex; BLG, a milk protein; KAP4-2, a protein component of wool; COL1α1, a bone protein used for taxonomic identification. Selected properties influencing protein detectability using LC–MS/MS techniques include predicted peptide size following trypsin digestion (cut sites are shown as dashed lines) and location of disulfide linkages (green boxes). Peptide lengths of 6–30 amino acids are most suitable for detection, (165) and those outside this range are marked as less likely to be found due to size (red border). Note that endogenous proteolysis of amelogenin during dental development causes additional cleavages that are not shown. (603) Only half (736 amino acids) of the collagen protein is shown for space reasons. (B) Comparison of the number of characterized species to the number of protein entries in UniProtKB (SwissProt and TrEMBL) for the major taxonomic classes Magnoliopsida (flowering plants), Mammalia (mammals), Aves (birds), and Actinopterygii (ray-finned fishes); inset shows enhanced view of the number of reviewed (SwissProt) protein entries. Numbers of characterized species were obtained from refs (604−607). (C) Reviewed and unreviewed protein entries available in UniProtKB for humans and common plants and animals consumed in ancient Mesoamerican diets; inset shows enhanced view of taxa with <1000 protein entries.

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Peptides from a given protein that are able to be consistently detected are known as proteotypic peptides; however, despite great effort, attempts to predict proteotypic peptides have been met with only limited success. (515−517) The inability to adequately correct for detection biases across the proteome represents a major challenge for protein quantitation (518) and is even more challenging for ancient samples and discovery-based applications.
One of the most vexing problems in shotgun proteomics is its limited reproducibility, especially for low abundance proteins, which are often the main proteins of interest in ancient protein studies. Two factors combine to contribute to this problem. First, gene expression, and hence protein abundance, typically varies by more than 6 orders of magnitude within a given tissue, (358,359,519) and even up to 12 orders of magnitude within biofluids. (520,521) This is starkly different from DNA, where most genes are present in a single copy and even multicopy genes rarely differ in abundance by more than 1 order of magnitude. (522) Thus, the dynamic range of a proteome is enormous. Second, current tandem mass spectrometers are not powerful enough to selectively fragment and analyze all precursor ions generated from a given sample, (514) and thus only a fraction of the complex peptide mixture is accessible by conventional shotgun proteomics. (523) For a given mass-to-charge (m/z) window, current instruments either select the most abundant ion for fragmentation and MS2 (DDA) or they select all ions, producing a mixed MS2 (DIA). (204) Neither are satisfying solutions for ancient proteins. DDA leads to poorly reproducible ion selection and underselection of low abundance peptides, while DIA produces MS2s that are difficult to deconvolute and still suffer from low signal-to-noise ratios for low abundance peptides. Targeting methods, such as MRM or PRM, only partially address the problem and are wasteful of sample, as they only measure preselected peptides, (524) which can be difficult to know in advance for discovery-based studies or when diagenetic modifications of target proteins are unknown. Better chromatography, ion mobility, faster, more powerful instruments, and computational improvements may soon ease some of these problems, (191,525−529) but at present ancient protein studies have yet to achieve the high degree of reproducibility that paleogenomics enjoys.

4.2. Protein Identification

Protein identification, especially from complex mixtures of unknown composition, remains an ongoing challenge. Sequence-based identification remains the gold standard in paleoproteomics, but such identification is strongly influenced by the choice of database and anticipated modifications, meaning that only peptides that are looked for can be identified. In the light of these limitations, some analysis strategies include error-tolerant or de novo sequencing approaches in order to increase the number of identified MS2 spectra beyond that of the database. However, as the sample search space increases through the addition of sequences or permitted modifications, computational demands can quickly exceed current feasibility. Fortunately, unlike detection limits, identification limitations are soft in that data can be later reanalyzed with updated software, algorithms, databases, computing infrastructure, and chemical modification parameters to improve identifications.
Databases are currently a major limiting factor in ancient protein identification. Although the proteomes of humans (359) and some model organisms (519,530,531) are now well curated and annotated, the proteomes of many taxa of archaeological interest, from molluscs to microbiomes, remain insufficiently or poorly characterized. Most databases suffer from inclusion bias, with model organisms and economically important taxa being vastly overrepresented compared to other species. Domesticated taurine cattle (Bos taurus), for example, has a published genome and a well-annotated proteome, with 140,740 entries in UniProtKB and 9,936,498 in NCBI GenBank. In contrast, only limited genetic sequence data are available for the banteng (Bos javanicus), a related species of cattle from Southeast Asia for which there are fewer than 400 protein sequences in NCBI GenBank and UniProtKB. Even in cases where a full genome for a species is available, there can be significant delays between when genomic data is submitted to a database, such as NCBI GenBank, and when its annotated proteome becomes available across linked platforms, such as UniProtKB. For example, at the time of writing, UniProtKB contained only 97 reviewed and 2,985 unreviewed proteins for domesticated water buffalo (Bubalus bubalis), but its entire genome was available through NCBI GenBank, including 64,378 annotated proteins. In addition, although UniProtKB aggregates data from major genetic databases, it does not incorporate all annotated genomic data, and some curated protein databases continue to be independent, such as the HOMD, WormBase, ParaSite, and VectorBase. Thus, protein representation even within large protein databases, such as UniProtKB, is highly variable across major taxonomic groups, especially when accounting for the number of known species per group (Figure 4B), and careful consideration must be given to database selection and database biases during study design.
Several commonly observed data artifacts in paleoproteomics studies are database-dependent and can be avoided by accounting for the known biases of specific databases and search algorithms. For example, where there is a substantial imbalance in the amount of protein sequence coverage for related taxa, database searching may incorrectly identify the protein as originating from the wrong species due to the higher number of conserved peptide matches to the better covered species, which may have more complete sequences as well as more isoforms. This is commonly observed in studies of domesticated bovids, which are frequently targeted for taxonomic discrimination using proteomic methods. Taurine cattle have roughly 1.5 times the amount of protein entries in UniProtKB than sheep (91,483) and goat (90,609) and the imbalance is even greater in NCBI GenBank, with taurine cattle having 20 times the amount of protein entries as sheep (336,178) and 40 times the amount of protein entries than goat (156,078). Proteomic analyses of these and other less well studied bovids generally return at least some protein taxonomic assignments to taurine cattle, the latter being an artifact of the more extensive representation of taurine cattle proteins in the database. While some taurine cattle protein entries in these databases are redundant, others contain important allelic sequence variants, and sequences generated from transcriptome data additionally include isoforms deriving from alternative splicing that are highly relevant for proteomics studies. When attempting to use proteomics to distinguish between closely related species, not only is the coverage of each species in the protein database important, so is the difference between the number of protein entries present.
Another common artifact occurs when databases contain entries for a protein with differing degrees of completeness, and entries containing only the mature protein sequence are preferentially (and sometimes incorrectly) identified over entries that also contain the signal peptides and other regions that are removed during mature protein formation. This has been observed in studies of mammalian collagen when search algorithms return spurious high-scoring assignments to extinct species, such as the giant South American ungulate Toxodon. In the case of Toxodon, the fossil COL1A1 protein entry in UniProtKB consists of only the helical region of the protein (UniProtKB C0HJP7, ∼1000 amino acids), whereas COL1A1 entries for extant taxa also include the flanking signal peptide, propeptide, and telopeptide regions, making it ∼500 amino acids longer. Signal and propeptides are removed during mature protein formation and are therefore never recovered from archaeological samples, while telopeptides are only rarely recovered. Because collagen is highly conserved across mammals, many recovered peptides will be shared among all ungulates, but these conserved peptides will have a higher percentage of protein coverage to Toxodon COL1A1 compared to extant taxa, making Toxodon a higher ranked match. Such artifacts can be easily identified and corrected by manually checking the sequence alignments and verifying the taxonomic specificity of the matches. Thus, sequence and taxonomic validation are important steps of data analysis in exploratory paleoproteomics studies.
Beyond issues of database completeness, databases also contain entries with varying levels of quality, annotation, and associated metadata. Within UniProtKB, the vast majority of sequences are unreviewed (TrEMBL), meaning that they are generated through an automated annotation process, while proteins whose annotations are manually reviewed (SwissProt) make up <0.5% of currently available protein data for flowering plants, mammals, birds, and fish (Figure 4B). While automatically annotated sequences in TrEMBL are largely accurate, they often require further review of the source entry and comparison with the same gene in other species. Sometimes it can be helpful to search against translated nucleotide databases (tblastn) in order to assess the range of species in which similar sequences have been identified. Many unreviewed entries also lack sufficient metadata for downstream analysis and may be simply annotated as a hypothetical or uncharacterized protein.
Collectively, these problems can be particularly acute for paleodietary studies in which ancient groups consumed a wide variety of foods that may or may not have adequate database representation. Consider, for example, the components of a hypothetical ancient Mesoamerican diet shown in Figure 4C, which includes common foods consumed in Central Mexico and the Maya lowlands. The human proteome is also included for comparison. At the time of writing, the number of total protein entries in UniProtKB for maize (Z. mays, 171,947) vastly exceeded that of other staple grains, such as amaranth (A. cruentus, 138), the database representation of white-tailed deer (O. virginianus, 37,513) and dwarfs that of red brocket deer (M. americana, 59). Protein entries for turkey (M. gallopavo, 17,051) greatly exceed those of muscovy duck (C. moschata, 146), as do entries for common beans (P. vulgaris, 32,845) compared to other legumes (L. esculenta, 9). Other food items, such as common vegetables (C. pepo, 667; D. ambrosioides, 124), local fish (A. felis, 37; M. urophthalmus, 65), and edible snails (L. esculenta, 9; P. flagellata, 4; P. indiorum, 1) have very few protein entries, effectively making them invisible in proteomic studies of ancient diets. Using an alternative database, such as NCBI RefSeq or GenBank, substantially improves the protein representation for some foods (e.g., C. pepo, 43,466) but not others (e.g., D. ambrosioides, 250; A. felis, 61; and P. indiorum, 1). Thus, while changing or combining databases can improve the identification of some genetically well-studied taxa, little can be done to improve the visibility of many other ancient Mesoamerican foods until more genetic data is available. Paleoproteomic characterization of ancient diets is therefore largely opportunistic. As such, while the identification of a dietary protein can be taken as positive evidence of its presence, the failure to identify a given food cannot be taken as evidence of its absence.
Beyond using existing protein databases, mining sequences from genetic and transcriptomic databases (proteogenomics) that are not automatically annotated by UniProtKB to create custom databases can increase identifications, as can de novo sequencing of proteins or peptides of interest. However, these options can be time-consuming and low-throughput if the proteins of interest are not already known or if the taxa themselves are understudied, which limits their feasibility in discovery-based applications. Massive gains in annotated genomic sequence data are needed to overcome this problem, and large-scale international efforts to dramatically increase the cataloging and characterization of eukaryotic biodiversity, such as the Earth BioGenome Project, (532) the Vertebrate Genomes Project, (533,534) and the Darwin Tree of Life Project, (535) offer great promise for improving the detection of dietary proteins in future paleodietary studies.
For now, however, even taxonomic identifications based on extremely well characterized proteins, such as COL1, face challenges. For example, there are currently no curated databases for COL1 PMF markers, which poses a large barrier to the exponentially growing ZooMS community. Beyond the taxonomic biases of proteomic databases, such as UniProtKB, and especially the underrepresentation of fish and birds, COL1 genes are often incorrectly translated from genetic data or contain incorrect annotations. In the absence of sufficient databases, multivariate analysis and other statistical tools have been successful at clustering COL1 and keratin peptide groups for taxonomic identification of sample types such as bone and hair. (315,536) Moreover, in the case of COL1 the charge pairing in the molecule required for fibril formation results in highly conserved tryptic cleavage sites, meaning that it is possible to compute all potential variants for every arginine- and lysine-terminated peptide, generating theoretically huge databases of 10222 collagen sequences, (20) and thereby potentially, albeit impractically, allowing the de novo identification of COL1 peptides with high fidelity from MS2 spectra.
As the field of paleoproteomics progresses, more consistent reporting of search settings and the percentage of identified spectra will help to highlight the extent of the problem of identification, as well as allow metaanalysis of the success of different search strategies for different types of samples. With the exponential growth of genomes, transcriptomes, and proteomes, increasing characterization of modifications, and development of new PMF markers, reanalysis of previous samples will likely result in an increasing amount of detected proteins and/or taxonomic resolution. However, this requires publication of raw data to facilitate reanalysis, as is already recommended for ancient LC–MS/MS data (4) and facilitated by the ProteomeXchange consortium (537) via repositories such as PRIDE. (538) As no global repository exists for MALDI-TOF data, it is currently less common for PMF raw data to be published, although growing numbers of data sets are found on the open-access general purpose repository Zenodo (https://zenodo.org/search?q=zooms), which was developed under the European OpenAIRE program and is run by CERN.

4.3. Protein Authentication

One challenge that particularly affects paleoproteomics is the difficulty of distinguishing authentically ancient peptides, proteins, and proteomes from environmental and modern contamination.

4.3.1. Sources of Contamination

Potential sources of contamination are myriad, but for modern proteomics they are mostly lab-derived and easily mitigated. Contamination poses a much more challenging problem for paleoproteomics, where the proteins of interest are degraded, in low abundance, and frequently unknown. Sources of contamination from the laboratory are well described (135) and can be identified and controlled through the analysis of extraction and instrument blanks and the application of best practices. (4)
Less controllable are environmental contaminants that are introduced over time by the burial or depositional context. Environmental contamination can be accounted for to some degree by collecting and analyzing control samples from other associated remains or nearby sediment or soil, although this is not always feasible. The inclusion of controls in the experimental design is particularly important for paleoproteomic studies on: (1) open systems, such as the identification of a nursing female dog from milk proteins found in rib bones; (539) (2) sediments or soils, such as the identification of silk from textile imprints in the soil; (332) and (3) infectious pathogens, especially taxa that have close environmental relatives, such as Mycobacterium tuberculosis. (437,475)
Even more variable and often unpredictable are contaminants introduced during excavation, handling, storage, and conservation. This is a particular problem when analyzing museum collections where handling and curation history are not well documented. Contamination can occur during handling when collections are used for teaching or placed on display, and contaminants can also be unintentionally introduced through local storage conditions and may include proteins from bacteria, fungi, and rodent or insect pests, as well as treatments used in their control. (540) Object conservation is also a major source of contamination. Although today museums and conservators have synthetic options for consolidants, adhesives, and preservatives, natural products were almost exclusively used in the past, and some continue to be used as they provide certain benefits over synthetic materials. (541,542) Those most relevant for paleoproteomics research are collagen based glues, (543) fibroin used to repair silk, (544) and egg and milk based glazes and treatments, such as the application of egg whites and phytic acid as a flame retardant. (545) While the impact of conservation efforts on ancient DNA, stable isotope, and radiocarbon analyses has been extensively investigated, (546−548) their effect on paleoproteomics has been less systematically studied. Best practices involve avoiding samples where treatment or extensive handling has taken place.

4.3.2. Methods of Authentication

There are two main approaches to authenticating ancient proteins. The first involves the identification of damage patterns characteristic of ancient proteins. The second examines the broader context of the proteins, including its associated metaproteome and other corroborating lines of evidence.
Among ancient biomolecules, DNA undergoes regular and predictable forms of degradation that produce characteristic types of ancient DNA damage, (549,550) the most important of which are DNA fragmentation and cytosine deamination. (551) Their mechanisms of formation are well understood, and they are so consistent that they can be used to authenticate ancient DNA (552) and even distinguish ancient and modern DNA sequences in heavily contaminated samples. (553) Proteins also undergo processes of degradation, and methods have been proposed to authenticate ancient proteins or proteomes in cases where sufficient numbers of identified MS2s are available for statistical analysis (e.g., ref (554)). The biochemical and structural complexity of proteins, however, makes damage-related authentication of ancient proteins considerably more complicated than for ancient DNA.
For very ancient samples, hydrolysis is expected to cause protein fragmentation, resulting in a bias toward shorter enzymatic peptides, and an increased number of nontryptic cleavage sites (reviewed in (127)). Diagenetic amino acid modifications have also been proposed as markers of authenticity, but due to the increased biological complexity of proteins, other factors besides age can strongly influence the production or prevention modifications. (9,555) In particular, deamidation of asparagine and glutamine have been proposed as markers of authentication in both PMF and LC–MS/MS data. (275,365,554,556−558) Unlike deamination in aDNA, however, deamidation also occurs in vivo (559) and is strongly impacted by both local depositional chemistry (560) and choice of extraction method. (140,561) Therefore, its use as a reliable age-related indicator has been questioned. (555,560,562,563)
In MALDI-TOF applications, COL1 deamidation has been used as a relative age marker and proposed as a criterion to identify intrusive samples. In practice, however, this requires very large data sets, and even when data for large numbers of samples (>2,000) are available, the accuracy of seriating samples by relative age class is less than 50%. (560) For LC–MS/MS studies, deamidation has mostly been analyzed semiquantitatively at the metaproteome level (e.g., refs (14,92,197)) in order to show that deamidation is a top modification in the data set. Some studies have attempted to authenticate specific proteins using deamidation patterns, (101,439) but this requires a relatively large data set to perform statistical analysis, which is not always possible, (223) and even in cases where enough data is available, the results are not straightforward to interpret. (562) Nevertheless, proteins of ancient origin should exhibit evidence of diagenesis. Caution should be exercised if reports contain evidence of unexpected chemical behavior such as the lack of any degradation of proteins over extended periods of archaeological or geological time (564) or if unexpected proteins are detected that are commonly used in the same facility. (565) Future improvements in understanding and modeling protein diagenesis at specific amino acid sites may one day enable protein damage to be used more reliably and quantitatively as an age indicator. For now, however, it is most effectively used as a qualitative indicator of age in combination with other authentication approaches.
Contextual analysis focusing on the composition of the metaproteome as a whole, together with other corroborating lines of evidence, is currently the most robust authentication approach for ancient proteins. One important feature of proteins is that their expression is tissue specific, and thus the composition of the ancient metaproteome itself can be used to aid in its authentication. Different tissues and substrates, such as bone, dental calculus, artist materials, and pottery food crusts, are expected to each have a different and distinctive protein composition, and this is reflected empirically in ancient samples (Figure 5). Clear patterns are readily apparent. Bone is dominated by collagens and proteins involved in fibrilar organization and mineralization, as well as blood clotting factors and plasma proteins. (364) Dental calculus is enriched in collagen and proteins associated with the innate immune system (especially neutrophils), and also contains salivary amylase and occasional dietary proteins. (92,445) Artist materials, such as paints, tend to be dominated by a single protein source, such as egg, (137) and pottery food crusts are highly diverse but contain elevated levels of animal and plant proteins associated with foods, in this case foods independently known to have been consumed at the site. (99) For sample types that naturally contain a microbial component, such as microbiomes, the bacterial protein and taxonomic composition can also serve as an authentication aid, (92,440,566) and for sample types that do not contain a microbial component during life or use, the relative proportions of microbial proteins to presumed endogenous proteins can also serve a relative preservation indicator.

Figure 5

Figure 5. Representative examples of ancient proteomes. Well-preserved ancient proteomes contain distinctive groups of proteins that reflect the protein composition of the original tissue or material, such as human bone (364) (A), human dental calculus (445) (B), artist materials (137) (C), and pottery crusts (99) (D). As such, the composition of an ancient proteome can aid in its authentication. Data were searched against the SwissProt database using Mascot using the parameters described in ref (102). Protein identifications were established at <5.0% protein FDR and <1.0% peptide FDR in Scaffold v.5 (Proteome Software), and proteins with a minimum of 97% protein identification probability and at least two unique peptides were accepted. The top 15 proteins (by number of PSMs) per sample source were visualized as a treemap and labeled by their corresponding gene name; trypsin, keratins, serum albumin, and microbial proteins were excluded from the analysis. *Ovostatin; **riboflavin-binding protein; ***B3-hordein.

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As more modern and ancient metaproteomes are characterized and their data made public, the characteristic features and composition of well-preserved metaproteomes can be defined and used as benchmarks for paleoproteomic authentication. Such data, in combination with independent corroborating lines of evidence, such as paleogenomic data, paleoethnobotanical evidence, or zooarchaeological findings, can be used to assist in establishing the plausibility of findings, particularly those that are extraordinary or unanticipated.

5. Future Directions: Taking on the Dark Proteome

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Despite significant technological advances, much of the ancient proteome still remains “dark” as seen in the consistently low percentage of identified MS2 spectra. This darkness, however, also represents an opportunity, as it means that there are still ancient proteins and proteomes to be discovered if only the right tools can be developed to access them. On the basis of current knowledge, the dark proteome can be divided into three types: (1) “structurally dark” regions of proteins (567) that are intrinsically disordered, never resolved, or (in the case of bottom up proteomics) lacking suitable cleavage sites; (2) “overlooked” peptides, peptides that have been modified to such a degree that their spectra are not recognizable by conventional MS2 analysis; and (3) “unidentifiable” protein fragments that have cross-linked or condensed into novel chimeric structures that have not been previously studied. Below we discuss new technologies that may assist with understanding and accessing the dark proteome, as well as consider the potential of emerging nonmass spectrometry-based sequencing approaches in paleoproteomics.

5.1. Emerging Technologies in Mass Spectrometry

Many of the unmatched queries found in ancient protein data sets are presumably the result of poor ion selection, fragmentation, and/or detection. A range of new instrumentation options are being developed that include faster data acquisition, higher sensitivity, and increased ion transfer efficiency. However, mass spectrometers are now reaching the point where chromatography will need to be improved in order for these instrument improvements to actually provide better data, (191) and yet paleoproteomic samples often suffer from poor chromatography due to unknown coextracted compounds or other factors. Alternatively, chromatography may be augmented or even eventually replaced by enhanced gas phase separation and fractionation, such as ion mobility, as is currently being implemented in Direct Infusion Shotgun Proteome Analysis (DISPA). (568) Future research development needs to focus on specific optimization for paleoproteomic samples, with the aim of improving precursor separation, reducing peak width, and generating better MS2 spectra. This will ultimately improve peptide identification and even enable de novo sequencing to resolve some of the overlooked peptides. This can be combined with improvements in databases, characterization of proteins, structural determination (e.g., AlphaFold (569)), and digestion (168) to aid in the recovery of structurally dark sequences.
Although chromatography improvements and database expansions represent low hanging fruit, identifying overlooked proteins and novel chimeric structures will be much more difficult, and it will require a better understanding of the diagenetic processes that facilitate preservation but inhibit recovery and challenge identification. In addition to side chain modification, backbone hydrolysis, and cyclization, peptides can undergo condensation with other molecules in the archaeological environment, including other proteins, lipids, carbohydrates, nucleic acids, metabolites, and inorganic compounds. Some of these reaction types are already targets for methodological improvement due to their presence in vivo (e.g., glycoproteins and some types of protein–protein and protein-nucleotide cross-linking). (509) However, many are likely novel degradation products or uncharacterized interactions that involve hydrolysis and reformation of the peptide backbone to form truly chimeric proteins.
One approach to this problem is to use experimental time series data to attempt to recreate the process of diagenesis. A time series has the advantage that even if compounds cannot be directly identified, their rise and fall highlights their place in a complex diagenetic pathway. Thankfully, the technologies to analyze these are routine rather than emerging. Raman spectroscopy can give insights into chemical character, amino acid analysis can detect the building blocks, and FT-ICR MS can establish the atomic composition of the masses. Similar approaches are used to understand the humification of soil organic matter (570,571) and the formation of melanoidins, the condensation products of proteins and carbohydrates. Greater structural insights into these diagenetic products will likely reveal a multiplicity of products each present in vanishingly small concentrations much like the hydrocarbon “hump” (unresolved complex mixtures of diagenetic components found in crude oils (572)) or the myriad of compounds identified in humic extracts.
An alternative approach is to visualize the interactions between the different combinations of peptides, proteins, and mineral surfaces that play a role in protecting sequences over deep time, but also likely inhibit recovery. Atomic force microscopy and high resolution 2D electron microscopy and 3D tomography imaging have already begun to inform the intimate association between, and therefore survival mechanisms of, collagen and mineral in bone. (573) Continued investigation into bone, as well as other biominerals and degradation products that produce mineral surfaces, will allow for both a better understanding of protein survival and, more importantly, modifications unique to such protein mineral interactions. Building a greater understanding of these modifications into search strategies will hopefully uncover more peptides, while pattern based discrimination analyses (e.g., machine learning) will assist in identifying groups of new products forming over time.
Spatial subcellular proteomics is still in its infancy, (574) but will likely make considerable advances over the next decade. (575) It combines super-resolution microscopy and top-down proteomic analysis to enable in situ mass spectrometry imaging (MSI). Spatial resolution will enhance our understanding of layered and otherwise structurally organized proteins such as cross sections of artwork or incremental precipitation of biologically induced or mediated mineral formation. The best known and highest resolution method is MALDI MSI, which is already providing detailed molecular images of tissues, but it is limited by its need for surface matrix deposition in many cases. Although their resolution is not as good as MALDI MSI, instruments which do not require matrix deposition and may be better suited to the size of paleoproteomic samples are being explored such as in situ DESI, nano-DESI, and LESA MSI. (194,195,576) Although current super-resolution microscopy approaches rely on fluorescence based methods (577) which are challenging for ancient samples, this is a growing field of research with new approaches being proposed and developed (e.g., Akoya Bioscience’s CODEX, Resolve’s Molecular Cartography combined with Zeiss Microscopes) which push the limits of imaging. Even if such imaging approaches are not suited for ancient samples, they can be used to investigate experimental samples to better characterize “overlooked” and “unidentifiable” parts of the dark proteome.

5.2. Beyond Mass Spectrometry?

For the past two decades, mass spectrometry has been the workhorse of proteomics and protein-based studies; nevertheless, mass spectrometry is also limited in that “instead of truly sequencing, it classifies a protein and typically requires about a billion copies of a protein to do it”. (67) A variety of other non-MS based technologies are currently emerging that offer the promise of both higher sensitivity and greater scale, such as massively parallel sequencing by nanopore, fluorosequencing, and image-based Edman. (67,68,578) At present, these techniques are not ideally suited to paleoproteomics due to the anticipated and unexpected modifications present in ancient samples, (579) as well as the autofluorescence caused by diagenesis, (580) but as these technologies continue to be optimized, their use may come within reach of paleoproteomics. In addition, the increasing focus on MS-based spatial and single cell proteomics mean that these technologies are far from the only options that will become available over the next decade as alternatives to conventional mass spectrometry.

6. Conclusion

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The mass spectrometry revolution in paleoproteomics has contributed to unprecedented findings over the past two decades, from the discovery of new archaic human fossils (184,291,292) to the detailed characterization of Neolithic and Bronze Age cuisines. (95,99,101,102,107,108,223,445) Ancient proteins have been particularly valuable for revealing ephemeral aspects of the past, such as the care and repair of art and museum objects, (509) the construction methods of books, (156,295) and even the difficulties of labor and childbirth. (432) While much of the ancient protein research over the past two decades has been exploratory and opportunistic in nature, the data produced and the knowledge gained will allow for larger scale and more directed questions to be explored in the coming decades. With this expansion, paleoproteomics is poised to provide insights from the past that can inform on matters ranging from ecological conservation to human health and wellbeing. Exciting new instruments and capabilities are on the horizon that promise to push the limits of our detection even farther, but only a better fundamental understanding of how proteins both degrade and persist will allow us to access the dark proteome that predominates in most archaeological samples. The past 20 years have been characterized by unprecedented gains in the ability to detect and identify ancient proteins, proteomes, and metaproteomes through advances in mass spectrometry. The next 20 years will surely hold many surprises as we begin to apply this analytical power to answer long-standing questions about the past and innovate new solutions to old problems.

Special Issue Paper

This paper is an additional review for Chem. Rev. 2021, volume 121, issue 19, “Frontiers of Analytical Science”.

Author Information

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  • Corresponding Author
    • Christina Warinner - Department of Anthropology, Harvard University, Cambridge, Massachusetts 02138, United States;  Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany;  Orcidhttps://orcid.org/0000-0002-4528-5877;  Email: [email protected]
  • Authors
    • Kristine Korzow Richter - Department of Anthropology, Harvard University, Cambridge, Massachusetts 02138, United States
    • Matthew J. Collins - Department of Archaeology, Cambridge University, Cambridge CB2 3DZ, United Kingdom;  Section for Evolutionary Genomics, Globe Institute, University of Copenhagen, Copenhagen 1350, Denmark;  Orcidhttps://orcid.org/0000-0003-4226-5501
  • Notes
    The authors declare no competing financial interest.

Biographies

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Christina Warinner is Associate Professor of Anthropology at Harvard University and Group Leader of Microbiome Sciences at the Max Planck Institute for Evolutionary Anthropology. She earned her Ph.D. from Harvard University in 2010 and received postdoctoral training in genomics and proteomics at the University of Zurich (2010-2012) and the University of Oklahoma (2012-2014). She specializes in the analysis of degraded DNA and proteins, and she led the first metagenomic and metaproteomic investigations of dental calculus. Her research focuses on the study of ancient biomolecules to better understand past human diet, health, and the evolution of the human microbiome, and she has made important contributions to understanding the prehistory of dairying, tracing the evolution of the oral microbiome, and reconstructing the migrations and cultural exchange networks of past human societies.

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Kristine Korzow Richter is a Research Associate in the Warinner Lab at Harvard University. She earned her Ph.D. from the Pennsylvania State University in 2014 before completing postdoctoral training in proteomics at the University of York (2015–2018) and the Max Planck Institute for the Science of Human History (2018–2020). She specializes in the preservation, recovery, and identification of ancient proteins for the taxonomic identification of animals. Her research focuses on the study of animal–human interactions including exploitation and management as well as ecosystem reconstruction.

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Matthew Collins is a Professor of Biomolecular Archaeology at the Globe Institute of the University of Copenhagen and the MacDonald Chair of Palaeoproteomics at the University of Cambridge. He was awarded a Ph.D. from the University of Glasgow (UCNW) in 1996 and received training in Chemistry and Biochemistry at the University of Leiden (1996–1991) and in microbiology and organic geochemistry at the University of Bristol (1991–1993) before taking a lectureship in Environmental Biogeochemistry at the University of Newcastle. He then moved to the University of York in 2003 to establish BioArCh, a center integrating Biology, Archaeology and Chemistry. In 2016, he was appointed a DNRF Niels Bohr Professor at the University of Copenhagen and the MacDonald Chair of Palaeoproteomics at the University of Cambridge. His research explores the survival of biomolecules, notably proteins in the archaeological and geological record, and the potential to use these to estimate the age and authenticity of samples in an attempt to recover biologically and archaeologically relevant information. A particular interest of his has been the potential of well-dated historical materials, notably parchment and wax, to explore past production processes from farm to factory.

Acknowledgments

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The authors thank Ashley Scott for her assistance with Figure 5, the Peabody Museum of Archaeology and Ethnology and Richard Meadow for allowing the PMF analysis of the faunal bone shown in Figure 3A, and two anonymous reviewers for providing valuable suggestions to improve the manuscript. This research was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement ERC-2017-StG 804844-DAIRYCULTURES to C.W. and ERC-2017-ADG 787282-Beasts to Craft to M.C.), the Werner Siemens Foundation (Paleobiotechnology) to C.W., the Danish National Research Foundation DNRF128 to M.C., the Max Planck Harvard Center for the Archaeoscience of the Ancient Mediterranean (MHAAM), and the Max Planck Society.

Glossary of Abbreviations and Acronyms

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AAR

amino acid racemization; the chiral conversion of l-amino acids into d-amino acids

AHSG

α2-HS glycoprotein, also known as fetuin-A; a protein that contributes to biomineralization

AMBN

ameloblastin; a major protein in enamel

AMBP

α-1-Microglobulin/Bikunin Precursor; a protein that has been detected in pottery residues

AMELX

X-chromosome isoform of the amelogenin protein; a major protein in enamel

AMELY

Y-chromosome isoform of the amelogenin protein; a major protein in enamel

AMTN

amelotin; a major protein in enamel

ASPN

asporin; a protein that facilitates tooth attachment to the periodontal ligament

AVD

avidin; a protein present in egg white

b ions

in protein tandem mass spectrometry, b ions are a series of fragment ions that extend from the N-terminus. Low energy collision induced dissociation (CID) typically produces pairs of b ions and y ions by breaking the peptide amide bond.

BGN

biglycan; a protein that facilitates biomineralization

BLASTp

protein–protein basic local alignment search tool; a tool that identifies regions of local similarity between protein sequences; it can be used to infer functional and evolutionary relationships

BLG

β-lactoglobulin; a major protein in the whey fraction of milk

bp

base pairs of double-stranded DNA

BP

Before Present, a standard unit of time used in radiocarbon dating that is calculated as radiocarbon years before 1950. May be uncalibrated (radiocarbon years before present, RCYBP) or calibrated (cal BP). For date estimations obtained without radiometric methods, the units Ka (kiloannuum, thousand years ago) or Ma (megaannuum, million years ago) are recommended.

CERN

Conseil Européen pour la Recherche Nucléaire; the European Organization for Nuclear Research

CID

collision-induced dissociation; a method of precursor ion fragmentation in tandem mass spectrometry that is widely used in proteomics and which primarily produces b and y ions

C-terminus

left-to-right nomenclature of an amino acid chain, referring to the last amino acid in the chain that has a free carboxylic acid group.

C3

complement component 3; a protein of the innate immune system that plays a key role in the complement system

C18

octadecyl carbon chain (C18)-bonded silica; used for protein and peptide purification

CBP

corneous β-proteins, formerly known as β-keratins; a group of structural proteins that are the predominant proteins in the hard corneous material of avian and reptilian scales, claws, beaks, and feathers and turtle shells

CD14

cluster of differentiation 14; a protein of the innate immune system predominantly produced by macrophages that binds bacterial lipopolysaccharide (LPS)

CDS

coding DNA sequence; the portion of a gene that is expressed into protein

CHAD

chondroadherin; a cartilage-associated protein expressed on bone articular surfaces

CHCA

α-cyano-4-hydroxycinnamic acid; a commonly used matrix in MALDI-TOF MS

CLU

clusterin; a widely expressed secretory glycoprotein in mammals, and also in bird eggshell and egg white

COL1

Type I collagen; the most abundant form of collagen in animals

COL12

Type XII collagen; a collagen protein that is found in association with type I collagen

cRAP

Common Repository of Adventitious Proteins; a list of common contaminants in mass spectrometry laboratories

CSN1S1

α S1 casein; a major milk protein

CSN2

β casein; a major milk protein

CTSG

cathepsin G; a defensive protein produced by cells of the innate immune system, especially neutrophils

d-amino acid

stereoisomeric form of an amino acid in the d-configuration (dextrorotatory, rotates polarized light rightwards). Although present in peptidoglycan and produced by bacteria, d-amino acids contribute minimally to the proteins of most living organisms. Most d-amino acids are believed to form through diagenetic racemization.

Da, kDa

dalton, kilodalton; a dalton is defined as one twelfth the mass of a free neutral atom of 12C at rest

DDA

data-dependent acquisition; a mode of data collection in tandem mass spectrometry in which the most intense precursor ions in a first stage of tandem mass spectrometry are then fragmented and analyzed in a second stage of tandem mass spectrometry

DDJB

DNA Databank of Japan; one of three consortium members in the INSDC

DEFA

defensin α 1, also known as neutrophil defensin 1; a cytotoxic protein produced by cells of the innate immune system, especially neutrophils

DEJ

dentine–enamel junction. The junction between the enamel crown and the underlying tooth dentine; a key site of enamel production during development.

DESI

desorption electrospray ionization

DHB

2,5-dihydroxybenzoic acid

DIA

data-independent acquisition; a mode of data collection in tandem mass spectrometry in which all precursor ions within a narrow m/z window in a first stage of tandem mass spectrometry are then fragmented in a second stage of mass spectrometry

DISPA

direct infusion shotgun proteome analysis

DNA

deoxyribonucleic acid

DPT

dermatopontin; an extracellular matrix protein that accelerates collagen fibril formation and stabilizes collagen fibrils

EDTA

ethylenediaminetetraacetic acid (EDTA), notable for its 2+ cation chelating ability. Widely used in ancient biomolecular studies to demineralize skeletal remains.

ELISA

enzyme-linked immunosorbent assay; a solid-phase type of immunoassay that can detect protein ligands in solution using antibodies

EMBL-EBI

European Bioinformatics Institute, based in Hinxton, UK; a component of the European Molecular Biology Laboratory, an intragovernmental organization headquartered in Heidelberg, Germany. One of three consortium members in the INSDC and a member of the UniProt Consortium

ENAM

enamelin; a major protein in enamel

Ensembl

a project run by EMBL-EBI that imports primary data from genome and genetic data archive resources and provides annotation of transcript structures, genomic variants, and regulatory regions

ESI

electrospray ionization; a form of soft ionization used by LC–MS/MS systems

EVA

ethylene-vinyl acetate; an elastomeric polymer used for minimally invasive protein sampling

F2

coagulation factor II, also known as prothrombin; a protein involved in blood coagulation

F7

coagulation factor VII; a protein involved in blood coagulation

F9

coagulation factor IX; a protein involved in blood coagulation

F10

coagulation factor X; a protein involved in blood coagulation

FASP

filter-aided sample preparation; a method used for protein extraction

FDR

False Discovery Rate; a statistical method for estimating type I errors. FDR-controlling procedures are applied to peptide and protein identifications to minimize spurious results

FT-ICR MS

Fourier-transform ion cyclotron mass spectrometry

GAPB

glyceraldehyde-3-phosphate dehydrogenase; a ubiquitous enzyme involved in glycolysis

GASP

Gel-Aided Sample Preparation; a method used for protein extraction

GenBank

genetic sequence database containing an annotated collection of all publicly available DNA sequences maintained by NCBI

Glu-C

endoproteinase that preferentially cleaves peptide bonds C-terminal to glutamic acid residues; also known as V-8 protease

GLYCAM1

glycosylation-dependent cell adhesion molecule-1; a mucin-like glycoprotein present in milk

HBA1

hemoglobin subunit α 1; a major component of hemoglobin in blood

HBBF

fetal hemoglobin subunit beta; a protein differentially expressed in the months before and after birth; relevant for studies animal skins and parchments

HBV

Hepatitis B virus

HOMD

Human Oral Microbiome Database; a curated online database of human oral microbes and associated genomic data and metadata developed and maintained by the Forsyth Institute

HPLC

high performance liquid chromatography; form of chromatographic separation widely used in protein tandem mass spectrometry workflows

HSP90A

inducible cytosolic isoform of heat shock protein 90; a protein differentially expressed in the months before and after birth. Relevant for studies animal skins and parchments.

INSDC

International Nucleotide Sequence Database Collaboration; a global body operated by the EMBL-EBI, NCBI, and DDJB that coordinates the storage and sharing of genetic sequence data, alignments, assemblies, and functional annotations

Ka

kiloannuum; thousand years ago

KAP4-2

keratin associated protein 4–2; a protein component of wool

kDa

unit of mass corresponding to 1000 Da

α-keratins

Alpha-keratins are a group of structural proteins that are the predominant proteins of vertebrate hair/fur, nails/claws, horns, hooves, quills, and baleen; a minor component of skin

KLK4

kallikrein related peptidase 4; an enamel protein

KRT75

Keratin type II cytoskeletal 75; a keratin protein that has been identified within enamel

l-amino acid

stereoisomeric form of an amino acid in the l-configuration (laevorotatory, rotates polarized light leftwards). With few exceptions, proteins within living organisms are made up of L-amino acids.

LC–MS/MS

liquid chromatography tandem mass spectrometry

LEGK

legumin K; an abundant protein in many plant seeds

LESA-MSI

liquid extraction surface analysis mass spectrometry imaging

LPO

lactoperoxidase; a defensive enzyme secreted by mammary and other mucosal glands

LRG1

leucine rich α-2-glycoprotein 1; a secreted glycoprotein of the innate immune system

LUM

lumican; a protein that supports collagen fibril organization

Lys-C

endoproteinase that cleaves peptide bonds C-terminal to lysine residues

Lys-N

metalloendoprotease that cleaves peptide bonds N-terminal to lysine residues

LYZ

lysozyme; an antimicrobial enzyme that is part of the innate immune system

Ma

megaannuum; million years ago

MALDI-MSI

matrix-assisted laser desorption/ionization mass spectrometry imaging

MALDI-TOF

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MGP

matrix gla protein; regulates biomineralization

MMP20

matrix metalloproteinase-20, also known as enamelysin; an enamel protein

MPO

myeloperoxidase; a prevalent protein in dental calculus that is produced by cells of the innate immune system, especially neutrophils

MRM

multiple reaction monitoring; a method that can be used in the targeted acquisition of tandem mass spectrometry data

MS1

first mass scan in tandem mass spectrometry

MS2

second mass scan in tandem mass spectrometry

MSI

mass spectrometry imaging

MUC5B

mucin 5B; a major gel-forming mucin in saliva and other mucus

m/z

mass-to-charge ratio

N-terminus

left-to-right nomenclature of an amino acid chain, referring to the first amino acid in the chain that has a free amine group

NCBI

National Center for Biotechnology Information; a governmental agency based in Bethesda, USA, that develops and coordinates information technology, databases, and software to support research in molecular biology, biochemistry, and genetics. One of three consortium members in the INSDC, it maintains the databases GenBank and RefSeq, among other resources

NCP

noncollagenous protein; refers to the noncollagenous proteins present within predominantly collagenous tissues, such as bone

ODAM

odontogeneic ameloblast-associated protein; a major protein in enamel

OIH

ovoinhibitor; antimicrobial protease inhibitor found in egg white and egg yolk

PCA

principal components analysis

PMF

peptide mass fingerprinting

PIP

prolactin induced protein; a protein involved in immunological function and fluid production

PIR

Protein Information Resource; a UniProt Consortium member

POSTN

periostin; a protein highly expressed in bone periosteum

PRIDE

PRoteomics IDEntifications (PRIDE) database; a major data repository of mass spectrometry-based proteomics data operated by EMBL-EBI

PRM

parallel reaction monitoring; a method that can be used in the targeted acquisition of tandem mass spectrometry data

ProAlanase

endoprotease that preferentially cleaves peptide bonds C-terminal to proline and, to a lesser extent, alanine

PSD

postsource decay

PTM

post-translational modification

PVC

polyvinyl chloride, a synthetic polymer of plastic; used in minimally destructive sampling protocols to obtain protein through the triboelectric effect

R group

a functional group within a molecule that has distinctive chemical properties; the R group of an amino acid determines which amino acid it is

RefSeq

a database of nonredundant annotated sequences representing genomic data, transcripts and proteins maintained by the NCBI

RIA

radioimmunoassay

RNA

ribonucleic acid

RPN2

ribophorin II; a protein expressed in the rough endoplasmic reticulum; relevant for studies animal skins and parchments

S100A8

S100 Calcium Binding Protein A8; a protein involved in the regulation of inflammation and immune response

S100A9

S100 Calcium Binding Protein A9; a protein involved in the regulation of inflammation and immune response

SDS

sodium dodecyl sulfate; a surfactant used during protein extraction

SERPINA1

Serpin Family A Member 1, also known as α-1-antitrypsin; a serine protease inhibitor

SERPINB14

ovalbumin; a storage protein that is the most abundant protein in egg white

SERPINF1

Serpin Family F Member 1; neurotrophic protein, also inhibits angiogenesis

SIB

Swiss Bioinformatics Institute; a member of the UniProt Consortium

SP3

single-pot, solid-phase-enhanced sample preparation; a method used for protein extraction

SPIN

species by proteome investigation; a DIA workflow for identifying mammalian species using tandem mass spectrometry

SPINK7

Serine Peptidase Inhibitor Kazal Type 7, also known as ovomucoid; an abundant serine peptidase inhibitor in egg white

SRM

selected reaction monitoring; a synonym for multiple reaction monitoring (MRM)

SwissProt

manually annotated and nonredundant protein sequence database component of the UniProtKB; maintained by the Swiss Institute of Bioinformatics (SIB)

TENP

transiently expressed in neural precursors, also known as BPI fold-containing family B, member 2 and ovoglobulin G2; a major protein in egg white

TF

transferrin; an iron binding transport protein

TfsA

tannerella surface protein A; a major component of the S-layer in Tannerella forsythia, a bacterium associated with dental plaque

TfsB

tannerella surface protein B; a major component of the S-layer in Tannerella forsythia, a bacterium associated with dental plaque

TOF

time-of-flight mass spectrometry

TrEMBL

translated EMBL Nucleotide Sequence Data Library; nonreviewed protein sequences translated from genetic data supplied by EMBL-EBI that have been computationally analyzed and enriched with automatic annotation and classification

UHPLC

ultra high performance liquid chromatography

UMOD

uromodulin; a glycoprotein produced by mammalian kidneys, abundant in urine

UniProtKB

UniProt Knowledgebase; centralized resource for protein metadata, including annotated structural and functional information; maintained by the UniProt consortium, which consists of the EMBL-EBI, SIB, and PIR

VIM

vimentin; a major cytoskeletal protein in mesenchymal cells

VTN

vitronectin; a cell adhesion protein found in serum and tissues

y ions

in protein tandem mass spectrometry, y ions are a series of fragment ions that extend from the C-terminus. Low energy collision induced dissociation (CID) typically produces pairs of b ions and y ions by breaking the peptide amide bond.

ZooMS

Zooarchaeology by Mass Spectrometry; an application of MALDI-TOF collagen PMF for taxonomic identification

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    Rybczynski Natalia; Gosse John C; Harington C Richard; Wogelius Roy A; Hidy Alan J; Buckley Mike
    Nature communications (2013), 4 (), 1550 ISSN:.
    The mid-Pliocene was a global warm period, preceding the onset of Quaternary glaciations. Here we use cosmogenic nuclide dating to show that a fossiliferous terrestrial deposit that includes subfossil trees and the northern-most evidence of Pliocene ice wedge casts in Canada's High Arctic (Ellesmere Island, Nunavut) was deposited during the mid-Pliocene warm period. The age estimates correspond to a general maximum in high latitude mean winter season insolation, consistent with the presence of a rich, boreal-type forest. Moreover, we report that these deposits have yielded the first evidence of a High Arctic camel, identified using collagen fingerprinting of a fragmentary fossil limb bone. Camels originated in North America and dispersed to Eurasia via the Bering Isthmus, an ephemeral land bridge linking Alaska and Russia. The results suggest that the evolutionary history of modern camels can be traced back to a lineage of giant camels that was well established in a forested Arctic.
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  11. 11
    van der Valk, T.; Pečnerová, P.; Díez-Del-Molino, D.; Bergström, A.; Oppenheimer, J.; Hartmann, S.; Xenikoudakis, G.; Thomas, J. A.; Dehasque, M.; Sağlıcan, E. Million-Year-Old DNA Sheds Light on the Genomic History of Mammoths. Nature 2021, 591, 265– 269,  DOI: 10.1038/s41586-021-03224-9
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    11
    Million-year-old DNA sheds light on the genomic history of mammoths
    van der Valk, Tom; Pecnerova, Patricia; Diez-del-Molino, David; Bergstroem, Anders; Oppenheimer, Jonas; Hartmann, Stefanie; Xenikoudakis, Georgios; Thomas, Jessica A.; Dehasque, Marianne; Saglican, Ekin; Fidan, Fatma Rabia; Barnes, Ian; Liu, Shanlin; Somel, Mehmet; Heintzman, Peter D.; Nikolskiy, Pavel; Shapiro, Beth; Skoglund, Pontus; Hofreiter, Michael; Lister, Adrian M.; Goetherstroem, Anders; Dalen, Love
    Nature (London, United Kingdom) (2021), 591 (7849), 265-269CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Temporal genomic data hold great potential for studying evolutionary processes such as speciation. However, sampling across speciation events would, in many cases, require genomic time series that stretch well back into the Early Pleistocene subepoch. Although theor. models suggest that DNA should survive on this timescale1, the oldest genomic data recovered so far are from a horse specimen dated to 780-560 thousand years ago2. Here we report the recovery of genome-wide data from three mammoth specimens dating to the Early and Middle Pleistocene subepochs, two of which are more than one million years old. We find that two distinct mammoth lineages were present in eastern Siberia during the Early Pleistocene. One of these lineages gave rise to the woolly mammoth and the other represents a previously unrecognized lineage that was ancestral to the first mammoths to colonize North America. Our analyses reveal that the Columbian mammoth of North America traces its ancestry to a Middle Pleistocene hybridization between these two lineages, with roughly equal admixt. proportions. Finally, we show that the majority of protein-coding changes assocd. with cold adaptation in woolly mammoths were already present one million years ago. These findings highlight the potential of deep-time palaeogenomics to expand our understanding of speciation and long-term adaptive evolution.
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  12. 12
    Orlando, L.; Ginolhac, A.; Zhang, G.; Froese, D.; Albrechtsen, A.; Stiller, M.; Schubert, M.; Cappellini, E.; Petersen, B.; Moltke, I. Recalibrating Equus Evolution Using the Genome Sequence of an Early Middle Pleistocene Horse. Nature 2013, 499, 74– 78,  DOI: 10.1038/nature12323
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    12
    Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse
    Orlando, Ludovic; Ginolhac, Aurelien; Zhang, Guojie; Froese, Duane; Albrechtsen, Anders; Stiller, Mathias; Schubert, Mikkel; Cappellini, Enrico; Petersen, Bent; Moltke, Ida; Johnson, Philip L. F.; Fumagalli, Matteo; Vilstrup, Julia T.; Raghavan, Maanasa; Korneliussen, Thorfinn; Malaspinas, Anna-Sapfo; Vogt, Josef; Szklarczyk, Damian; Kelstrup, Christian D.; Vinther, Jakob; Dolocan, Andrei; Stenderup, Jesper; Velazquez, Amhed M. V.; Cahill, James; Rasmussen, Morten; Wang, Xiaoli; Min, Jiumeng; Zazula, Grant D.; Seguin-Orlando, Andaine; Mortensen, Cecilie; Magnussen, Kim; Thompson, John F.; Weinstock, Jacobo; Gregersen, Kristian; Roed, Knut H.; Eisenmann, Vera; Rubin, Carl J.; Miller, Donald C.; Antczak, Douglas F.; Bertelsen, Mads F.; Brunak, Soren; Al-Rasheid, Khaled A. S.; Ryder, Oliver; Andersson, Leif; Mundy, John; Krogh, Anders; Gilbert, M. Thomas P.; Kjaer, Kurt; Sicheritz-Ponten, Thomas; Jensen, Lars Juhl; Olsen, Jesper V.; Hofreiter, Michael; Nielsen, Rasmus; Shapiro, Beth; Wang, Jun; Willerslev, Eske
    Nature (London, United Kingdom) (2013), 499 (7456), 74-78CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The rich fossil record of equids has made them a model for evolutionary processes. Here the authors present a 1.12-times coverage draft genome from a horse bone recovered from permafrost dated to approx. 560-780 thousand years before present (kyr BP). These data represent the oldest full genome sequence detd. so far by almost an order of magnitude. For comparison, the authors sequenced the genome of a Late Pleistocene horse (43 kyr BP), and modern genomes of five domestic horse breeds (Equus ferus caballus), a Przewalski's horse (E. f. przewalskii) and a donkey (E. asinus). The analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0-4.5 million years before present (Myr BP), twice the conventionally accepted time to the most recent common ancestor of the genus Equus. In addn., the horse population size fluctuated multiple times over the past 2 Myr, particularly during periods of severe climatic changes. The authors est. that the Przewalski's and domestic horse populations diverged 38-72 kyr BP, and find no evidence of recent admixt. between the domestic horse breeds and the Przewalski's horse investigated. This supports the contention that Przewalski's horses represent the last surviving wild horse population. The authors find similar levels of genetic variation among Przewalski's and domestic populations, indicating that the former are genetically viable and worthy of conservation efforts. The authors also find evidence for continuous selection on the immune system and olfaction throughout horse evolution. Finally, the authors identify 29 genomic regions among horse breeds that deviate from neutrality and show low levels of genetic variation compared to the Przewalski's horse. Such regions could correspond to loci selected early during domestication.
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  13. 13
    Welker, F.; Ramos-Madrigal, J.; Kuhlwilm, M.; Liao, W.; Gutenbrunner, P.; de Manuel, M.; Samodova, D.; Mackie, M.; Allentoft, M. E.; Bacon, A.-M. Enamel Proteome Shows That Gigantopithecus Was an Early Diverging Pongine. Nature 2019, 576, 262– 265,  DOI: 10.1038/s41586-019-1728-8
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    13
    Enamel proteome shows that Gigantopithecus was an early diverging pongine
    Welker, Frido; Ramos-Madrigal, Jazmin; Kuhlwilm, Martin; Liao, Wei; Gutenbrunner, Petra; de Manuel, Marc; Samodova, Diana; Mackie, Meaghan; Allentoft, Morten E.; Bacon, Anne-Marie; Collins, Matthew J.; Cox, Jurgen; Lalueza-Fox, Carles; Olsen, Jesper V.; Demeter, Fabrice; Wang, Wei; Marques-Bonet, Tomas; Cappellini, Enrico
    Nature (London, United Kingdom) (2019), 576 (7786), 262-265CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch1. Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16-5.3 million years ago), remain unclear2,3. Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphol., the absence of cranial and post-cranial remains1,3-6, and the lack of independent mol. validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China7,8. The thermal age of these protein sequences is approx. five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12-10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addn., we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously obsd. in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus9,10. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic anal. into geog. areas and time periods previously considered incompatible with the preservation of substantial amts. of genetic information.
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  14. 14
    Cappellini, E.; Welker, F.; Pandolfi, L.; Ramos-Madrigal, J.; Samodova, D.; Rüther, P. L.; Fotakis, A. K.; Lyon, D.; Moreno-Mayar, J. V.; Bukhsianidze, M. Early Pleistocene Enamel Proteome from Dmanisi Resolves Stephanorhinus Phylogeny. Nature 2019, 574, 103– 107,  DOI: 10.1038/s41586-019-1555-y
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    14
    Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny
    Cappellini, Enrico; Welker, Frido; Pandolfi, Luca; Ramos-Madrigal, Jazmin; Samodova, Diana; Ruther, Patrick L.; Fotakis, Anna K.; Lyon, David; Moreno-Mayar, J. Victor; Bukhsianidze, Maia; Rakownikow Jersie-Christensen, Rosa; Mackie, Meaghan; Ginolhac, Aurelien; Ferring, Reid; Tappen, Martha; Palkopoulou, Eleftheria; Dickinson, Marc R.; Stafford, Jr, Thomas W.; Chan, Yvonne L.; Gotherstrom, Anders; Nathan, Senthilvel K. S. S.; Heintzman, Peter D.; Kapp, Joshua D.; Kirillova, Irina; Moodley, Yoshan; Agusti, Jordi; Kahlke, Ralf-Dietrich; Kiladze, Gocha; Martinez-Navarro, Bienvenido; Liu, Shanlin; Sandoval Velasco, Marcela; Sinding, Mikkel-Holger S.; Kelstrup, Christian D.; Allentoft, Morten E.; Orlando, Ludovic; Penkman, Kirsty; Shapiro, Beth; Rook, Lorenzo; Dalen, Love; Gilbert, M. Thomas P.; Olsen, Jesper V.; Lordkipanidze, David; Willerslev, Eske
    Nature (London, United Kingdom) (2019), 574 (7776), 103-107CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixt. events for extinct taxa1. However, the irreversible post-mortem degrdn.2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approx. 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approx. 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of mol. evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here, we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approx. 1.77-Myr old, recovered from the archaeol. site of Dmanisi (South Caucasus, Georgia)10. Mol. phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides addnl. information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of mol. evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.
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  15. 15
    Buckley, M.; Warwood, S.; van Dongen, B.; Kitchener, A. C.; Manning, P. L. A Fossil Protein Chimera; Difficulties in Discriminating Dinosaur Peptide Sequences from Modern Cross-Contamination. Proc. R. Soc. B: Biol. Sci. 2017, 284, 20170544,  DOI: 10.1098/rspb.2017.0544
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    15
    A fossil protein chimera; difficulties in discriminating dinosaur peptide sequences from modern cross-contamination
    Buckley, Michael; Warwood, Stacey; van Dongen, Bart; Kitchener, Andrew C.; Manning, Phillip L.
    Proceedings of the Royal Society B: Biological Sciences (2017), 284 (1855), 20170544/1-20170544/8CODEN: PRSBC7 ISSN:. (Royal Society)
    A decade ago, reports that org.-rich soft tissue survived from dinosaur fossils were apparently supported by proteomics-derived sequence information of exceptionally well-preserved bone. This initial claim to the sequencing of endogenous collagen peptides from an approx. 68 Myr Tyrannosaurus rex fossil was highly controversial, largely on the grounds of potential contamination from either bacterial biofilms or from lab. practice. In a subsequent study, collagen peptide sequences from an approx. 78 Myr Brachylophosaurus Canadensis fossil were reported that have remained largely unchallenged. However, the endogeneity of these sequences relies heavily on a single peptide sequence, apparently unique to both dinosaurs. Given the potential for cross-contamination from modern bone analyzed by the same team, here we ext. collagen from bone samples of three individuals of ostrich, Struthio camelus. The resulting LC-MS/MS data were found to match all of the proposed sequences for both the original Tyrannosaurus and Brachylophosaurus studies. Regardless of the true nature of the dinosaur peptides, our finding highlights the difficulty of differentiating such sequences with confidence. Our results not only imply that cross-contamination cannot be ruled out, but that appropriate measures to test for endogeneity should be further evaluated.
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  16. 16
    Cleland, T. P.; Schroeter, E. R.; Zamdborg, L.; Zheng, W.; Lee, J. E.; Tran, J. C.; Bern, M.; Duncan, M. B.; Lebleu, V. S.; Ahlf, D. R. Mass Spectrometry and Antibody-Based Characterization of Blood Vessels from Brachylophosaurus Canadensis. J. Proteome Res. 2015, 14, 5252– 5262,  DOI: 10.1021/acs.jproteome.5b00675
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    16
    Mass Spectrometry and Antibody-Based Characterization of Blood Vessels from Brachylophosaurus canadensis
    Cleland, Timothy P.; Schroeter, Elena R.; Zamdborg, Leonid; Zheng, Wenxia; Lee, Ji Eun; Tran, John C.; Bern, Marshall; Duncan, Michael B.; Lebleu, Valerie S.; Ahlf, Dorothy R.; Thomas, Paul M.; Kalluri, Raghu; Kelleher, Neil L.; Schweitzer, Mary H.
    Journal of Proteome Research (2015), 14 (12), 5252-5262CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Structures similar to blood vessels in location, morphol., flexibility, and transparency have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty osteonal network after degrdn. of original org. components. Here, we test the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resoln. mass spectrometry and confirmed by immunofluorescence. Two lines of evidence support this hypothesis. First, peptide sequencing of Brachylophosaurus canadensis blood vessel exts. is consistent with peptides comprising extant archosaurian blood vessels and is not consistent with a bacterial, cellular slime mold, or fungal origin. Second, proteins identified by mass spectrometry can be localized to the tissues using antibodies specific to these proteins, validating their identity. Data are available via ProteomeXchange with identifier PXD001738.
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  17. 17
    Schroeter, E. R.; DeHart, C. J.; Cleland, T. P.; Zheng, W.; Thomas, P. M.; Kelleher, N. L.; Bern, M.; Schweitzer, M. H. Expansion for the Brachylophosaurus Canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein. J. Proteome Res. 2017, 16, 920– 932,  DOI: 10.1021/acs.jproteome.6b00873
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    17
    Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein
    Schroeter, Elena R.; DeHart, Caroline J.; Cleland, Timothy P.; Zheng, Wenxia; Thomas, Paul M.; Kelleher, Neil L.; Bern, Marshall; Schweitzer, Mary H.
    Journal of Proteome Research (2017), 16 (2), 920-932CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Sequence data from biomols. such as DNA and proteins, which provide crit. information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontol. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiol., it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extn. methodol., high-resoln. mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamg. previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amt. of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.
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  18. 18
    Knoll, A. H. Paleobiological Perspectives on Early Microbial Evolution. Cold Spring Harb. Perspect. Biol. 2015, 7, a018093,  DOI: 10.1101/cshperspect.a018093
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    Baldauf, S. L.; Roger, A. J.; Wenk-Siefert, I.; Doolittle, W. F. A Kingdom-Level Phylogeny of Eukaryotes Based on Combined Protein Data. Science 2000, 290, 972– 977,  DOI: 10.1126/science.290.5493.972
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    19
    A kingdom-level phylogeny of eukaryotes based on combined protein data
    Baldauf, Sandra L.; Roger, A. J.; Wenk-Siefert, I.; Doolittle, W. F.
    Science (Washington, D. C.) (2000), 290 (5493), 972-977CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Current understanding of the higher order systematics of eukaryotes relies largely on analyses of the small ribosomal subunit RNA (SSU rRNA). Independent testing of these results is still limited. We have combined the sequences of four of the most broadly taxonomically sampled proteins available to create a roughly parallel data set to that of SSU rRNA. The resulting phylogenetic tree shows a no. of striking differences from SSU rRNA phylogeny, including strong support for most major groups and several major supergroups.
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  20. 20
    Welker, F.; Collins, M. J.; Thomas, J. A.; Wadsley, M.; Brace, S.; Cappellini, E.; Turvey, S. T.; Reguero, M.; Gelfo, J. N.; Kramarz, A. Ancient Proteins Resolve the Evolutionary History of Darwin’s South American Ungulates. Nature 2015, 522, 81– 84,  DOI: 10.1038/nature14249
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    20
    Ancient proteins resolve the evolutionary history of Darwin's South American ungulates
    Welker, Frido; Collins, Matthew J.; Thomas, Jessica A.; Wadsley, Marc; Brace, Selina; Cappellini, Enrico; Turvey, Samuel T.; Reguero, Marcelo; Gelfo, Javier N.; Kramarz, Alejandro; Burger, Joachim; Thomas-Oates, Jane; Ashford, David A.; Ashton, Peter D.; Rowsell, Keri; Porter, Duncan M.; Kessler, Benedikt; Fischer, Roman; Baessmann, Carsten; Kaspar, Stephanie; Olsen, Jesper V.; Kiley, Patrick; Elliott, James A.; Kelstrup, Christian D.; Mullin, Victoria; Hofreiter, Michael; Willerslev, Eske; Hublin, Jean-Jacques; Orlando, Ludovic; Barnes, Ian; MacPhee, Ross D. E.
    Nature (London, United Kingdom) (2015), 522 (7554), 81-84CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    No large group of recently extinct placental mammals remains as evolutionarily cryptic as the ∼280 genera grouped as South American native ungulates. To Charles Darwin, who first collected their remains, they included perhaps the strangest animal[s] ever discovered. Today, much like 180 yr ago, it is no clearer whether they had 1 origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million yr ago, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria. Morphol.-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degrdn. in semitropical and temperate deposits. Here we apply proteomic anal. to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain ∼90% direct sequence coverage of type I collagen α1- and α2-chains, representing ∼900 of 1140 amino-acid residues for each subunit. A phylogeny is estd. from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates from condylarths, a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and anal. procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time.
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  21. 21
    Horn, I. R.; Kenens, Y.; Palmblad, N. M.; van der Plas-Duivesteijn, S. J.; Langeveld, B. W.; Meijer, H. J. M.; Dalebout, H.; Marissen, R. J.; Fischer, A.; Vincent Florens, F. B. Palaeoproteomics of Bird Bones for Taxonomic Classification. Zool. J. Linn. Soc. 2019, 186, 650– 665,  DOI: 10.1093/zoolinnean/zlz012
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    There is no corresponding record for this reference.
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    Macek, B.; Forchhammer, K.; Hardouin, J.; Weber-Ban, E.; Grangeasse, C.; Mijakovic, I. Protein Post-Translational Modifications in Bacteria. Nat. Rev. Microbiol. 2019, 17, 651– 664,  DOI: 10.1038/s41579-019-0243-0
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    22
    Protein post-translational modifications in bacteria
    Macek, Boris; Forchhammer, Karl; Hardouin, Julie; Weber-Ban, Eilika; Grangeasse, Christophe; Mijakovic, Ivan
    Nature Reviews Microbiology (2019), 17 (11), 651-664CODEN: NRMACK; ISSN:1740-1526. (Nature Research)
    A review. Over the past decade the no. and variety of protein post-translational modifications that have been detected and characterized in bacteria have rapidly increased. Most post-translational protein modifications occur in a relatively low no. of bacterial proteins in comparison with eukaryotic proteins, and most of the modified proteins carry low, substoichiometric levels of modification; therefore, their structural and functional anal. is particularly challenging. The no. of modifying enzymes differs greatly among bacterial species, and the extent of the modified proteome strongly depends on environmental conditions. Nevertheless, evidence is rapidly accumulating that protein post-translational modifications have vital roles in various cellular processes such as protein synthesis and turnover, nitrogen metab., the cell cycle, dormancy, sporulation, spore germination, persistence and virulence. Further research of protein post-translational modifications will fill current gaps in the understanding of bacterial physiol. and open new avenues for treatment of infectious diseases.
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    Witze, E. S.; Old, W. M.; Resing, K. A.; Ahn, N. G. Mapping Protein Post-Translational Modifications with Mass Spectrometry. Nat. Methods 2007, 4, 798– 806,  DOI: 10.1038/nmeth1100
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    23
    Mapping protein post-translational modifications with mass spectrometry
    Witze, Eric S.; Old, William M.; Resing, Katheryn A.; Ahn, Natalie G.
    Nature Methods (2007), 4 (10), 798-806CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    A review. Post-translational modifications of proteins control many biol. processes, and examg. their diversity is crit. for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale expts. possible, screening complex mixts. of proteins for alterations in chem. modifications. By profiling protein chemistries, biologists can gain deeper insight into biol. control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.
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    Protein splicing and related forms of protein autoprocessing
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    Annual Review of Biochemistry (2000), 69 (), 447-496CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
    A review with 105 refs. Protein splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a protein, accompanied by the concomitant joining of the flanking polypeptide sequences, the exteins, by a peptide bond. It requires neither cofactors nor auxiliary enzymes and involves a series of four intramol. reactions, the first three of which occur at a single catalytic center of the intein. Protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools. Some of the reactions characteristic of protein splicing also occur in other forms of protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonprotein moieties. These mechanistic similarities may be the result of convergent evolution, but in at least one case-hedgehog protein autoprocessing-there is definitely a close evolutionary relationship to protein splicing.
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    Protein Language: Post-Translational Modifications Talking to Each Other
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    Trends in Plant Science (2018), 23 (12), 1068-1080CODEN: TPSCF9; ISSN:1360-1385. (Elsevier Ltd.)
    A Review Post-translational modifications (PTMs) are at the heart of many cellular signaling events. Apart from a single regulatory PTM, there are also PTMs that function in orchestrated manners. Such PTM crosstalk usually serves as a fine-tuning mechanism to adjust cellular responses to the slightest changes in the environment. While PTM crosstalk has been studied in depth in various species; in plants, this field is just emerging. In this review, we discuss recent studies on crosstalk between three of the most common protein PTMs in plant cells, being phosphorylation, ubiquitination, and sumoylation, and we highlight the diverse underlying mechanisms as well as signaling outputs of such crosstalk.
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    Protein homeostasis: live long, won't prosper
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    A review. Protein turnover is an effective way of maintaining a functional proteome, as old and potentially damaged polypeptides are destroyed and replaced by newly synthesized copies. An increasing no. of intracellular proteins, however, have been identified that evade this turnover process and instead are maintained over a cell's lifetime. This diverse group of long-lived proteins might be particularly prone to accumulation of damage and thus have a crucial role in the functional deterioration of key regulatory processes during ageing.
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    A short half-life of ULBP1 at the cell surface due to internalization and proteosomal degradation
    Fernandez-Messina, Lola; Reyburn, Hugh T.; Vales-Gomez, Mar
    Immunology & Cell Biology (2016), 94 (5), 479-485CODEN: ICBIEZ; ISSN:0818-9641. (NPG Nature Asia-Pacific)
    The expression of NKG2D ligands (NKG2D-L) flag stressed cells for immune recognition and destruction. A precise control of the cell surface expression of these proteins is therefore required to ensure an appropriate immune response and it is becoming clear that NKG2D ligand expression is regulated at multiple levels. We now report that the surface stability of the human glycosyl-phosphatidyl-inositol (GPI)-anchored ligand ULBP1 (UL16-binding protein) at the plasma membrane is lower than other ULBP mols. This difference in stability is due neither to shedding nor to a higher internalization rate of ULBP1 but rather occurs because of a rapid degrdn. of ULBP1 protein after internalization from the cell surface that is blocked by proteasome inhibition. These data indicate that, in addn. to the known transcriptional and post-translational mechanisms, surface expression of human NKG2D-L is also regulated by protein turnover and that the brief residence of ULBP1 could contribute to the fine tuning of immune responses.
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    Helfman, P. M.; Bada, J. L. Aspartic Acid Racemization in Tooth Enamel from Living Humans. Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 2891– 2894,  DOI: 10.1073/pnas.72.8.2891
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    Aspartic acid racemization in tooth enamel from living humans
    Helfman, Patricia M.; Bada, Jeffrey L.
    Proceedings of the National Academy of Sciences of the United States of America (1975), 72 (8), 2891-4CODEN: PNASA6; ISSN:0027-8424.
    The aspartic acid in human tooth enamel showed increasing racemization with increasing age. This increase was not seen in the metabolically active protein hemoglobin. The rate const. for the racemization reaction of aspartic acid in human tooth enamel was 8.29 × 10-4 year, suggesting that in any protein with a long in vivo lifetime, D-aspartic acid will accumulate with age (∼8% of total aspartic acid in enamel will be the D-enantiomer after 60 years). Thus, racemization may play some role in the aging process affecting metabolically stable tissues in long-lived homeotherms. Aspartic acid racemization in tooth enamel also provides a biochronol. tool for assessing the age of living mammals.
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    Stewart, D. N.; Lango, J.; Nambiar, K. P.; Falso, M. J. S.; FitzGerald, P. G.; Rocke, D. M.; Hammock, B. D.; Buchholz, B. A. Carbon Turnover in the Water-Soluble Protein of the Adult Human Lens. Mol. Vis. 2013, 19, 463– 475
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    Carbon turnover in the water-soluble protein of the adult human lens
    Stewart, Daniel N.; Lango, Jozsef; Nambiar, Krishnan P.; Falso, Miranda J. S.; FitzGerald, Paul G.; Rocke, David M.; Hammock, Bruce D.; Buchholz, Bruce A.
    Molecular Vision (2013), 19 (), 463-475CODEN: MVEPFB; ISSN:1090-0535. (Molecular Vision)
    Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the 14C bomb pulse. Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were sepd. into water-sol. and water-insol. fractions. The small mols. were removed using 3 kDa spin filters. The 14C/C was measured in paired protein fractions by accelerator mass spectrometry, and an av. age for the material within the sample was assigned using the 14C bomb pulse. The water-insol. fractions possessed 14C/C ratios consistent with the age of the cells. In all cases, the water-sol. fractions contained carbon that was younger than the paired water-insol. fraction. As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract.
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    Chemical and physical properties of old silk fabrics
    Becker, Mary A.; Magoshi, Yoshiko; Sakai, Tetsuya; Tuross, Noreen C.
    Studies in Conservation (1997), 42 (1), 27-37CODEN: SCONAH; ISSN:0039-3630. (International Institute for Conservation of Historic and Artistic Works)
    Amino acids recovered after std. hydrolysis are predictive indicators of the state of preservation of silk and provide a rapid method that can be applied to extremely small sample sizes. Biochem. data from 17 Japanese silk kimono lining fabrics are compared to previously reported data on occidental silks. The chem. and phys. data from old silk fabrics are used to illustrate the connection between information gained from a microgram of sample required for amino acids anal. and the state of preservation. Comparison of the Japanese and occidental silk fabrics also provides insight into the effects that different methods of caring for silk objects have on their longevity.
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    Nature (London, United Kingdom) (1953), 171 (), 737-8CODEN: NATUAS; ISSN:0028-0836.
    W. and C. propose a new structure for the Na salt of deoxyribose nucleic acid. This structure, which loosely resembles Furberg's model No. 1 (C.A. 47, 9924g), has 2 helical polynucleotide chains each coiled around the same axis but whose sequence of atoms runs in opposite directions. The chains are held together by H-bonding between purine and pyrimidine bases, a purine of 1 chain bonded to a pyrimidine of the other. Full details will be published elsewhere.
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    Central dogma of molecular biology
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    The history of the central dogma of mol. biol., which states that residue-by-residue transfer of sequential information cannot occur from protein to protein or to nucleic acid, is presented; and the 9 possible transfers are tentatively regrouped into 3 classes: general, special, and unknown transfers (those postulated by the central dogma). The general transfers, DNA → DNA (the arrow represents directional flow of detailed sequence information), DNA → RNA, and RNA → protein, are those that can occur in all cells. Special transfers, RNA → RNA, RNA → DNA, and DNA → protein, may occur in special circumstances. Unknown transfers, protein → protein, protein → DNA, protein → RNA, the central dogma postulates, never occur. The current validity of the central dogma is discussed.
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    Amino acid racemization reactions and their geochemical implications
    Bada, Jeffrey L.; Schroeder, Roy A.
    Naturwissenschaften (1975), 62 (2), 71-9CODEN: NATWAY; ISSN:0028-1042.
    A review is presented with 44 refs.
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    Bada, J. L.; Gillespie, R.; Gowlett, J. A.; Hedges, R. E. Accelerator Mass Spectrometry Radiocarbon Ages of Amino Acid Extracts from Californian Palaeoindian Skeletons. Nature 1984, 312, 442– 444,  DOI: 10.1038/312442a0
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    Accelerator mass spectrometry radiocarbon ages of amino acid extracts from Californian paleoindian skeletons
    Bada, J. L.; Gillespie, R.; Gowlett, J. A. J.; Hedges, R. E. M.
    Nature (London, United Kingdom) (1984), 312 (5993), 442-4CODEN: NATUAS; ISSN:0028-0836.
    Accelerator mass spectrometry radiocarbon dating of amino acids in paleoindian skeletal remains from California was performed and the results compared with ages detd. from aspartic acid racemization rates. The radiocarbon ages detd. by using the total amino acid exts. correlated well with convention radiocarbon ages using the collagen component, and both techniques indicated that the skeletons were of Holocene age. Aspartic acid racemization ages thus appear to be too old, in which Upper Pleistocene age of 17,150 yr before present was assigned. Radiocarbon dating by accelerator mass spectroscopy also enables an accurate detn. of amino acid racemization rates.
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    Bada, J. L.; Schroeder, R. A.; Carter, G. F. New Evidence for the Antiquity of Man in North America Deduced from Aspartic Acid Racemization. Science 1974, 184, 791– 793,  DOI: 10.1126/science.184.4138.791
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    New evidence for the antiquity of man in North America deduced from aspartic acid racemization
    Bada, Jeffrey L.; Schroeder, Roy A.; Carter, George F.
    Science (Washington, DC, United States) (1974), 184 (4138), 791-3CODEN: SCIEAS; ISSN:0036-8075.
    Ages of several California Paleo-Indian skeletons were deduced from the extent of aspartic acid racemization. Apparently man was present in North America at ≤50,000 years before the present.
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    Wehmiller, J. F. Interlaboratory Comparison of Amino Acid Enantiomeric Ratios in Fossil Pleistocene Mollusks. Quat. Res. 1984, 22, 109– 120,  DOI: 10.1016/0033-5894(84)90010-3
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    Interlaboratory comparison of amino acid enantiomeric ratios in fossil Pleistocene mollusks
    Wehmiller, John F.
    Quaternary Research (1984), 22 (1), 109-20CODEN: QRESAV; ISSN:0033-5894.
    Enantiomer ratios (D/L) were detd. by 11 labs. for 8 amino acids occurring in 3 homogeneous powd. samples of fossil Pleistocene mollusks. Analyses were made by 3 gas chromatog. methods and by 1 type of ion-exchange chromatog. Coeffs. of variation were (1) 3-8% for alanine, glutamic acid, and aspartic acid; (2) 5-10% for leucine and phenylalanine; and (3) 10-18% for isoleucine, proline, and valine. Anal. precision for individual labs. was usually 2-5%, but significant differences were often encountered between the results of different labs.; in some cases these differences would have led to very large differences (25% or more) in age ests. based on enantiomer ratios. Multiple analyses of desalted hydrolyzates of the powd. samples suggest that the interlab. differences are often caused by instrumental and/or derivatization procedures, rather than the wet chem. techniques of sample prepn. It is proposed that these powd. samples be used as a ref. materials for all future detns. of amino acid enantiomer ratios in fossils.
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    Wehmiller, J. F.; York, L. L.; Bart, M. L. Amino Acid Racemization Geochronology of Reworked Quaternary Mollusks on U.S. Atlantic Coast Beaches: Implications for Chronostratigraphy, Taphonomy, and Coastal Sediment Transport. Mar. Geol. 1995, 124, 303– 337,  DOI: 10.1016/0025-3227(95)00047-3
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    Amino acid racemization geochronology of reworked Quaternary mollusks on U.S. Atlantic coast beaches: implications for chronostratigraphy, taphonomy, and coastal sediment transport
    Wehmiller, John F.; York, Linda L.; Bart, Michelle L.
    Marine Geology (1995), 124 (1/4), 303-37CODEN: MAGEA6; ISSN:0025-3227. (Elsevier)
    Approx. 200 shells (primarily Mercenaria) from 21 beach sites between New Jersey and Florida have been analyzed for the extent of racemization (epimerization) of their fossilized amino acids. The greatest concn. of sites is in North Carolina, in regions least affected by human modification of natural beach sediment-transport processes. These results can be used to est. the frequency of age mixing of Pleistocene shell material in Holocene depositional environments. Selected shells have also been dated by 14C (conventional and/or AMS) to provide direct calibration of the amino acid epimerization ratios. Shell taphonomic characteristics (particularly color) can be qual. related to apparent ages. Data for shell fragmentation, combined with amino acid age ests., provide insights into probable transport distances of reworked shells. Shells with apparent Pleistocene ages have epimerization values equiv. to those seen in onshore exposures of Pleistocene coastal units. Amino acid epimerization measurements are a comparatively rapid and inexpensive chronol. tool for use in taphonomic and sediment-(shell-) transport studies. Paired amino acid-14C analyses on single shells provide insights into geochem. alteration of shells and permit modeling of the residence time of shells on beaches. Greater nos. of analyses at each site are needed before statistically valid ests. of age mixing can be obtained, but the apparent distribution of Pleistocene shells on Atlantic coast beaches appears to be related to the distribution of Pleistocene units in the shoreface and inner shelf, and the thickness of Holocene sedimentary cover in specific areas. These observations provide useful criteria for the evaluation of possible age mixing of shells collected at outcrops of Pleistocene units in the Coastal Plain.
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    Dickinson, M. R.; Lister, A. M.; Penkman, K. E. H. A New Method for Enamel Amino Acid Racemization Dating: A Closed System Approach. Quat. Geochronol. 2019, 50, 29– 46,  DOI: 10.1016/j.quageo.2018.11.005
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    Demarchi, B.; Williams, M. G.; Milner, N.; Russell, N.; Bailey, G.; Penkman, K. Amino Acid Racemization Dating of Marine Shells: A Mound of Possibilities. Quat. Int. 2011, 239, 114– 124,  DOI: 10.1016/j.quaint.2010.05.029
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    Amino acid racemization dating of marine shells: A mound of possibilities
    Demarchi Beatrice; Williams Matt G; Milner Nicky; Russell Nicola; Bailey Geoff; Penkman Kirsty
    Quaternary international : the journal of the International Union for Quaternary Research (2011), 239 (1-2), 114-124 ISSN:1040-6182.
    Shell middens are one of the most important and widespread indicators for human exploitation of marine resources and occupation of coastal environments. Establishing an accurate and reliable chronology for these deposits has fundamental implications for understanding the patterns of human evolution and dispersal. This paper explores the potential application of a new methodology of amino acid racemization (AAR) dating of shell middens and describes a simple protocol to test the suitability of different molluscan species. This protocol provides a preliminary test for the presence of an intracrystalline fraction of proteins (by bleaching experiments and subsequent heating at high temperature), checking the closed system behaviour of this fraction during diagenesis. Only species which pass both tests can be considered suitable for further studies to obtain reliable age information. This amino acid geochronological technique is also applied to midden deposits at two latitudinal extremes: Northern Scotland and the Southern Red Sea. Results obtained in this study indicate that the application of this new method of AAR dating of shells has the potential to aid the geochronological investigation of shell mounds in different areas of the world.
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    Kaufman, D. Dating Deep-Lake Sediments by Using Amino Acid Racemization in Fossil Ostracodes. Geology 2003, 31, 1049– 1052,  DOI: 10.1130/G20004.1
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    Dating deep-lake sediments by using amino acid racemization in fossil ostracodes
    Kaufman, Darrell
    Geology (2003), 31 (12), 1049-1052CODEN: GLGYBA; ISSN:0091-7613. (Geological Society of America)
    The long-term rate of racemization for amino acids preserved in fossil ostracod shells was detd. using independently dated sediment cores from 5 deep lakes. The racemization rates for aspartic and glutamic acids in the common ostracod genus Candona were calibrated for the past 100 Kyr, providing the basis for an age equation with a realistic age uncertainty of ∼±20%. The new age equation can be applied to other hypolimnic settings because the temp. below the thermocline of deep lakes from middle- and high-latitude regions can reasonably be assumed to remain close to 4°. The sample size required for anal. is 1 order of magnitude less than for 14C dating and presents new opportunities to date deposits that are org.-matter poor or those with large 14C reservoir effects.
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    West, G.; Kaufman, D. S.; Muschitiello, F.; Forwick, M.; Matthiessen, J.; Wollenburg, J.; O’Regan, M. Amino Acid Racemization in Quaternary Foraminifera from the Yermak Plateau, Arctic Ocean. Geochronol. 2019, 1, 53– 67,  DOI: 10.5194/gchron-1-53-2019
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    THE ELECTRON MICROSCOPY OF RANCHO LA BREA BONE
    WYCKOFF R W; DOBERENZ A R
    Proceedings of the National Academy of Sciences of the United States of America (1965), 53 (), 230-3 ISSN:0027-8424.
    There is no expanded citation for this reference.
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    Wyckoff, R. W.; Mccaughey, W. F.; Doberenz, A. R. The Amino Acid Composition of Proteins from Pleistocene Bones. Biochim. Biophys. Acta 1964, 93, 374– 377,  DOI: 10.1016/0304-4165(64)90387-3
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    The amino acid composition of proteins from pleistocene bones
    Wyckoff, Ralph W. G.; McCaughey, William F.; Doberenz, Alexander R.
    Biochimica et Biophysica Acta, General Subjects (1964), 93 (2), 374-7CODEN: BBGSB3; ISSN:0304-4165.
    Complete amino acid analyses have been made of proteins persisting in 3 pleistocene bones from the Rancho La Brea tar pits and in a recent desert-dried bone used as control. The total amt. of protein in the fossils lies between 1/6 and 1/2 that in the recent bone. The ratios of amino acids in the 4 samples are similar and point to the presence of other proteins besides the collagen which the electron microscope shows to be present.
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    Akiyama, M.; Wyckoff, R. W. The Total Amino Acid Content of Fossil Pecten Shells. Proc. Natl. Acad. Sci. U. S. A. 1970, 67, 1097– 1100,  DOI: 10.1073/pnas.67.3.1097
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    Total amino acid content of fossil pecten shells
    Akiyama, Masahiko; Wyckoff, Ralph W. G.
    Proceedings of the National Academy of Sciences of the United States of America (1970), 67 (3), 1097-100CODEN: PNASA6; ISSN:0027-8424.
    Amino acid anal. have been made of the insol. protein, sol. peptide, and free amino acid fractions isolated from a series of fossil pecten shells of ages from the Pleistocene through the Jurassic. The total amino acid content declines progressively with age of the fossil but, in contrast to oyster shells, only a small fraction of the initial protein is present in the Pleistocene and older specimens. Though the thermally less stable acids are depleted in the free acid fraction, this is not true for the insol. proteins and peptides. Large amts. of free amino acids and peptides persist through the Pliocene, considerable quantities of peptide have been recovered from the Miocene specimens, and even the oldest fossils have retained analyzable amts. of all 3 fractions.
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    Miller, M. F., 2nd; Wyckoff, R. W. Proteins in Dinosaur Bones. Proc. Natl. Acad. Sci. U. S. A. 1968, 60, 176– 178,  DOI: 10.1073/pnas.60.1.176
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    Proteins in dinosaur bones
    Miller, Mahlon F., II; Wyckoff, Ralph W. G.
    Proceedings of the National Academy of Sciences of the United States of America (1968), 60 (1), 176-8CODEN: PNASA6; ISSN:0027-8424.
    Proteins contg. 20 or more amino acids were recovered from individual bones 150 million years old. Preliminary results were given and the possible origin of the proteins was discussed.
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    Doberenz, A. R.; Miller, M. F., 2nd; Wyckoff, R. W. An Analysis of Fossil Enamel Protein. Calcif. Tissue Res. 1969, 3, 93– 95,  DOI: 10.1007/BF02058649
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    Analysis of fossil enamel protein
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    Calcified Tissue Research (1969), 3 (1), 93-5CODEN: CATRBZ; ISSN:0008-0594.
    An anal. was made of the enamel protein of a mastodon molar tooth. The enamel resembled fresh bovine enamel. A significant amt. of hydroxyproline was present in the residual enamel. The data indicated that hydroxyproline is present in the enamel protein and not as contamination from the dentin.
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    de Jong, E. W.; Westbroek, P.; Westbroek, J. W.; Bruning, J. W. Preservation of Antigenic Properties of Macromolecules over 70 Myr. Nature 1974, 252, 63– 64,  DOI: 10.1038/252063a0
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    Preservation of antigenic properties of macromolecules over 70 Myr
    de Jong E W; Westbroek P; Westbroek J W; Bruning J W
    Nature (1974), 252 (5478), 63-4 ISSN:0028-0836.
    There is no expanded citation for this reference.
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    Lowenstein, J. M. Immunological Reactions from Fossil Material. Philos. Trans. R. Soc. London B Biol. Sci. 1981, 292, 143– 149,  DOI: 10.1098/rstb.1981.0022
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    Immunological reactions from fossil material
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    Philosophical transactions of the Royal Society of London. Series B, Biological sciences (1981), 292 (1057), 143-9 ISSN:0962-8436.
    Genetic relations among living species can be deduced from biochemical as well as morphological similarities, but our understanding of fossil species has depended entirely on their morphology. Residual proteins in fossils might provide genetic information, but their small quantity and chemical alterations due to time and environmental agents have prevented the obtaining of species-specific analysis. This report describes a radioimmunoassay capable of detecting extremely small amounts of fossil proteins, such as collagen and albumin. Species-specific proteins have been identified in a frozen Siberian mammoth, a Pleistocene bison, and a series of human fossils that includes Neanderthal, Homo erectus and Australopithecus robustus. This technique promises to provide molecular data on the genetic affinities of fossil and living species.
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    Lowenstein, J. M.; Sarich, V. M.; Richardson, B. J. Albumin Systematics of the Extinct Mammoth and Tasmanian Wolf. Nature 1981, 291, 409– 411,  DOI: 10.1038/291409a0
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    Albumin systematics of the extinct mammoth and Tasmanian wolf
    Lowenstein J M; Sarich V M; Richardson B J
    Nature (1981), 291 (5814), 409-11 ISSN:0028-0836.
    There is no expanded citation for this reference.
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    Muyzer, G.; Westbroek, P.; De Vrind, J. P. M.; Tanke, J.; Vrijheid, T.; De Jong, E. W.; Bruning, J. W.; Wehmiller, J. F. Immunology and Organic Geochemistry. Org. Geochem. 1984, 6, 847– 855,  DOI: 10.1016/0146-6380(84)90107-4
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    Immunology and organic geochemistry
    Muyzer, G.; Westbroek, P.; De Vrind, J. P. M.; Tanke, J.; Vrijheid, T.; De Jong, E. W.; Bruning, J. W.; Wehmiller, J. F.
    Organic Geochemistry (1984), 6 (Adv. Org. Geochem. 1983), 847-55CODEN: ORGEDE; ISSN:0146-6380.
    Two conventional antibody prepns. directed against fractions of sol. macromols. from shells of the recent bivalve Ensis ensis were allowed to react with shell fragments of a large variety of invertebrates, mainly mollusks. Use was made of a special adaptation of the ELISA. One antibody prepn. aE1, reacted with a broad variety of taxa, including representatives of nonmolluskan phyla, whereas the other, aE3, only recognized determinants in taxa closely related to Ensis. Reactions with fossils were obtained with both antisera. By incubation of antibody solns. with etched shell powders of nonEnsis shells more specific prepns. were obtained that gave more meaningful systematic results. In an artificial diagenesis expt. carried out with shell fragments of Mercenaria mercenaria the degrdn. of individual determinants of the sol. matrix could be followed with monoclonal antibodies. These data are related to the amino acid racemization profile. In the light of the available information, the possible significance of immunol. for org. geochem. research is discussed.
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    Ulrich, M. M.; Perizonius, W. R.; Spoor, C. F.; Sandberg, P.; Vermeer, C. Extraction of Osteocalcin from Fossil Bones and Teeth. Biochem. Biophys. Res. Commun. 1987, 149, 712– 719,  DOI: 10.1016/0006-291X(87)90426-8
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    Extraction of osteocalcin from fossil bones and teeth
    Ulrich, M. M. W.; Perizonius, W. R. K.; Spoor, C. F.; Sandberg, P.; Vermeer, C.
    Biochemical and Biophysical Research Communications (1987), 149 (2), 712-19CODEN: BBRCA9; ISSN:0006-291X.
    Osteocalcin (also called bone Gla-protein) was detected in fossil bovid bones ranging from 12,000 to 13 million yr old and in rodent teeth 30 million yr old. Both the antigenic activity and the protein-bound Gla-residues have remained intact. The protein is indistinguishable from recent bovine osteocalcin when analyzed by HPLC using ion-exchange and size-exclusion columns. If sufficient amts. can be extd. and an adequate purifn. procedure is established, this would be the 1st time that amino acid sequences in a protein from fossil bones may be detd. Such sequence data could offer a new approach to the phylogenetic study of extinct taxa.
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    Smith, P. R.; Wilson, M. T. Detection of Haemoglobin in Human Skeletal Remains by ELISA. J. Archaeol. Sci. 1990, 17, 255– 268,  DOI: 10.1016/0305-4403(90)90023-X
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    Collins, M. J.; Muyzer, G.; Westbroek, P.; Curry, G. B.; Sandberg, P. A.; Xu, S. J.; Quinn, R.; Mackinnon, D. Preservation of Fossil Biopolymeric Structures: Conclusive Immunological Evidence. Geochim. Cosmochim. Acta 1991, 55, 2253– 2257,  DOI: 10.1016/0016-7037(91)90101-A
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    Preservation of fossil biopolymeric structures: Conclusive immunological evidence
    Collins, M. J.; Muyzer, G.; Westbroek, P.; Curry, G. B.; Sandberg, P. A.; Xu, S. J.; Quinn, R.; MacKinnon, D.
    Geochimica et Cosmochimica Acta (1991), 55 (8), 2253-7CODEN: GCACAK; ISSN:0016-7037.
    Immunol. reactions previously reported with exts. from biominerals at least 70 Myr old have fuelled hopes that such biopolymeric structures can survive the process of fossilization. Antibodies were raised against biopolymers from a range of recent brachiopod shells to reconstruct a fine-grained pattern of phylogenetic relationships. Using the same antibodies, the phylogenetic affinities of fossil relatives were tested. Immunol. reaction patterns for Plio-Pleistocene shells were essentially identical to the patterns of modern shells (although the breadth of reactivity was narrowed) and entirely consistent with current systematic interpretations. Older samples (>4-21 Myr) were immunol. reactive, but failed to satisfy the criterion of systematic specificity. The results provide an unequivocal demonstration that original macromol. structures capable of yielding systematic information are preserved and accessible for immunol. anal. for at least two million years. Nonspecific reactions obsd. with the older material may relate in part to formation of mineral-induced diagenetic determinants.
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    Brandt, E.; Wiechmann, I.; Grupe, G. How Reliable Are Immunological Tools for the Detection of Ancient Proteins in Fossil Bones?. Int. J. Osteoarchaeol. 2002, 12, 307– 316,  DOI: 10.1002/oa.624
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    Leach, J. D. A Brief Comment on the Immunological Identification of Plant Residues on Prehistoric Stone Tools and Ceramics: Results of a Blind Test. J. Archaeol. Sci. 1998, 25, 171– 175,  DOI: 10.1006/jasc.1997.0237
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    Schweitzer, M.; Hill, C. L.; Asara, J. M.; Lane, W. S.; Pincus, S. H. Identification of Immunoreactive Material in Mammoth Fossils. J. Mol. Evol. 2002, 55, 696– 705,  DOI: 10.1007/s00239-002-2365-6
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    Identification of immunoreactive material in mammoth fossils
    Schweitzer, Mary Higby; Hill, Christopher L.; Asara, John M.; Lane, William S.; Pincus, Seth H.
    Journal of Molecular Evolution (2002), 55 (6), 696-705CODEN: JMEVAU; ISSN:0022-2844. (Springer-Verlag New York Inc.)
    A study was conducted to demonstrate that exts. of fossils themselves can be used to produce antibodies that are fossil specific. Materials extd. from a well-preserved 100,000-300,000 yr-old mammoth skull was used to produce antisera. The specificity of the antisera was tested by ELISA western blotting and immunohistochem. It was demonstrated that antisera reacted specifically with the fossils and not the surrounding sediments. Reactivity of antisera with modern proteins and tissues was also demonstrated, as well as the ability to detect evolutionary relationships via antibody-antigen interactions. Mass spectrometry demonstrated the presence of amino acids and specific peptides within the fossil. Peptides were purified by anion-exchange chromatog. and sequenced by tandem mass spectrometry. The collagen-derived peptides may have been the source of at least some of the immunol. reactivity, but the antisera identified mols. that were not obsd. by mass spectrometry, indicating that immunol. methods may have greater sensitivity.
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    Huq, N. L.; Tseng, A.; Chapman, G. E. Partial Amino Acid Sequence of Osteocalcin from an Extinct Species of Ratite Bird. Biochem. Int. 1990, 21, 491– 496
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    Partial amino acid sequence of osteocalcin from an extinct species of ratite bird
    Huq, N. Laila; Tseng, Albert; Chapman, George E.
    Biochemistry International (1990), 21 (3), 491-6CODEN: BIINDF; ISSN:0158-5231.
    Osteocalcin the major γ-carboxyglutamic acid-contg. protein of vertebrate bone has been purified from the bones of a specimen of Pachyornis elephantopus, a species of the extinct class of New Zealand ratite birds, the moas. The sequence of the N-terminal region of moa osteocalcin was detd. using gas phase N-terminal sequencing. The N-terminal sequences of the ostrich and the rhea osteocalcins were also detd. Alignment of the N-terminal sequence of osteocalcin from the extinct moa against the osteocalcins of the extant ostrich, rhea, and emu reveals the homol. among the ratite species is greater than the homol. with the chicken osteocalcin.
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    Smith, J. B. Peptide Sequencing by Edman Degradation. e LS 2001. .
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    Timp, W.; Timp, G. Beyond Mass Spectrometry, the Next Step in Proteomics. Sci. Adv. 2020, 6, eaax8978  DOI: 10.1126/sciadv.aax8978
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    Swaminathan, J.; Boulgakov, A. A.; Hernandez, E. T.; Bardo, A. M.; Bachman, J. L.; Marotta, J.; Johnson, A. M.; Anslyn, E. V.; Marcotte, E. M. Highly Parallel Single-Molecule Identification of Proteins in Zeptomole-Scale Mixtures. Nat. Biotechnol. 2018, 36, 1076– 1082,  DOI: 10.1038/nbt.4278
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    Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures
    Swaminathan, Jagannath; Boulgakov, Alexander A.; Hernandez, Erik T.; Bardo, Angela M.; Bachman, James L.; Marotta, Joseph; Johnson, Amber M.; Anslyn, Eric V.; Marcotte, Edward M.
    Nature Biotechnology (2018), 36 (11), 1076-1082CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
    The identification and quantification of proteins lags behind DNA-sequencing methods in scale, sensitivity, and dynamic range. Here, we show that sparse amino acid-sequence information can be obtained for individual protein mols. for thousands to millions of mols. in parallel. We demonstrate selective fluorescence labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor decreases in each mol.'s fluorescence after consecutive rounds of Edman degrdn. The obtained sparse fluorescent sequence of each mol. was then assigned to its parent protein in a ref. database. We tested the method on synthetic and naturally derived peptide mols. in zeptomole-scale quantities. We also fluorescently labeled phosphoserines and achieved single-mol. positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should enable studies of increasingly complex proteomic mixts., with the high sensitivity and digital quantification offered by single-mol. sequencing.
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    Ostrom, P. H.; Gandhi, H.; Strahler, J. R.; Walker, A. K.; Andrews, P. C.; Leykam, J.; Stafford, T. W.; Kelly, R. L.; Walker, D. N.; Buckley, M. Unraveling the Sequence and Structure of the Protein Osteocalcin from a 42ka Fossil Horse. Geochim. Cosmochim. Acta 2006, 70, 2034– 2044,  DOI: 10.1016/j.gca.2006.01.004
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    Unraveling the sequence and structure of the protein osteocalcin from a 42ka fossil horse
    Ostrom, Peggy H.; Gandhi, Hasand; Strahler, John R.; Walker, Angela K.; Andrews, Philip C.; Leykam, Joseph; Stafford, Thomas W.; Kelly, Robert L.; Walker, Danny N.; Buckley, Mike; Humpula, James
    Geochimica et Cosmochimica Acta (2006), 70 (8), 2034-2044CODEN: GCACAK; ISSN:0016-7037. (Elsevier)
    The authors report the first complete amino acid sequence and evidence of secondary structure for osteocalcin from a temperate fossil. The osteocalcin derives from a 42 ka equid bone excavated from Juniper Cave, Wyoming. Results were detd. by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS) and Edman sequencing with independent confirmation of the sequence in two labs. The ancient sequence was compared to that of three modern taxa: horse (Equus caballus), zebra (Equus grevyi), and donkey (Equus asinus). Although there was no difference in sequence among modern taxa, MALDI-MS and Edman sequencing show that residues 48 and 49 of our modern horse are Thr, Ala rather than Pro, Val as previously reported (B. Carstanjen et al., 2002). MALDI-MS and Edman sequencing data indicate that the osteocalcin sequence of the 42 kyr fossil is similar to that of modern horse. Previously inaccessible structural attributes for ancient osteocalcin were obsd. Glu39 rather than Gln39 is consistent with deamidation, a process known to occur during fossilization and aging. Two post-translational modifications were documented: Hyp9 and a disulfide bridge. The latter suggests at least partial retention of secondary structure. As has been done for ancient DNA research, the authors recommend stds. for prepn. and criteria for authenticating results of ancient protein sequencing.
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    Nielsen-Marsh, C. M.; Ostrom, P. H.; Gandhi, H.; Shapiro, B.; Cooper, A.; Hauschka, P. V.; Collins, M. J. Sequence Preservation of Osteocalcin Protein and Mitochondrial DNA in Bison Bones Older than 55 Ka. Geology 2002, 30, 1099– 1102,  DOI: 10.1130/0091-7613(2002)030<1099:SPOOPA>2.0.CO;2
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    Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older than 55 ka
    Nielsen-Marsh, Christina M.; Ostrom, Peggy H.; Gandhi, Hasand; Shapiro, Beth; Cooper, Alan; Hauschka, Peter V.; Collins, Matthew J.
    Geology (2002), 30 (12), 1099-1102CODEN: GLGYBA; ISSN:0091-7613. (Geological Society of America)
    The first complete sequence of the protein osteocalcin from small amts. of two bison bone (Bison priscus) dated older than 55.6 ka and older than 58.9 ka is reported. Osteocalcin was purified using new gravity columns followed by microbore reverse-phase high-performance liq. chromatog. Sequencing of osteocalcin employed two methods of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS); peptide mass mapping and post-source decay (PSD). PMM shows that ancient and modern bison osteocalcin have the same mass to charge (m/z) distribution, indicating an identical protein sequence and absence of diagenetic products. This was confirmed by PSG of the m/z 2066 tryptic peptide (residues 1-19); the mass spectra from ancient and modern peptides were identical. The 129 mass unit difference in the mol. ion between cow (bos taurus) and bison is caused by a single amino-acid substitution between the taxa. Bison mitochondrial control region DNA sequences were obtained from the older than 55.6 ka fossil. These results suggest that DNA and protein sequences can be used to directly investigate mol. phylogenies over a considerable time period, the abs. limit of which is yet to be detd.
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    Wu, C.; Duan, J.; Liu, T.; Smith, R. D.; Qian, W.-J. Contributions of Immunoaffinity Chromatography to Deep Proteome Profiling of Human Biofluids. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2016, 1021, 57– 68,  DOI: 10.1016/j.jchromb.2016.01.015
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    Contributions of immunoaffinity chromatography to deep proteome profiling of human biofluids
    Wu, Chaochao; Duan, Jicheng; Liu, Tao; Smith, Richard D.; Qian, Wei-Jun
    Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2016), 1021 (), 57-68CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
    A review. Human biofluids, esp. blood plasma or serum, hold great potential as the sources of candidate biomarkers for various diseases; however, the enormous dynamic range of protein concns. in biofluids represents a significant anal. challenge for detecting promising low-abundance proteins. Over the last decade, various immunoaffinity chromatog. methods have been developed and routinely applied for sepg. low-abundance proteins from the high- and moderate-abundance proteins, thus enabling much more effective detection of low-abundance proteins. Herein, we review the advances of immunoaffinity sepn. methods and their contributions to the proteomic applications in human biofluids. The limitations and future perspectives of immunoaffinity sepn. methods are also discussed.
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    Buckley, M.; Collins, M.; Thomas-Oates, J.; Wilson, J. C. Species Identification by Analysis of Bone Collagen Using Matrix-Assisted Laser Desorption/ionisation Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, 3843– 3854,  DOI: 10.1002/rcm.4316
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    Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry
    Buckley, Michael; Collins, Matthew; Thomas-Oates, Jane; Wilson, Julie C.
    Rapid Communications in Mass Spectrometry (2009), 23 (23), 3843-3854CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Species identification of fragmentary bone, such as in rendered meat and bone meal or from archaeol. sites, is often difficult in the absence of clear morphol. markers. Here we present a robust method of analyzing genus-specific collagen peptides by mass spectrometry simply by using solid-phase extn. (a C18 ZipTip) for peptide purifn., rather than liq. chromatog./mass spectrometry (LC/MS). Anal. of the collagen from 32 different mammal species identified a total of 92 peptide markers that could be used for species identification, for example, in processed food and animal feed. A set of ancient (>100 ka@10°) bone samples was also analyzed to show that the proposed method has applications to archaeol. bone identification. Copyright © 2009 John Wiley & Sons, Ltd. Dicerorhinus sumatrensis.
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    Buckley, M.; Whitcher Kansa, S.; Howard, S.; Campbell, S.; Thomas-Oates, J.; Collins, M. Distinguishing between Archaeological Sheep and Goat Bones Using a Single Collagen Peptide. J. Archaeol. Sci. 2010, 37, 13– 20,  DOI: 10.1016/j.jas.2009.08.020
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  74. 74
    Nielsen-Marsh, C. M.; Richards, M. P.; Hauschka, P. V.; Thomas-Oates, J. E.; Trinkaus, E.; Pettitt, P. B.; Karavanic, I.; Poinar, H.; Collins, M. J. Osteocalcin Protein Sequences of Neanderthals and Modern Primates. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 4409– 4413,  DOI: 10.1073/pnas.0500450102
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    74
    Osteocalcin protein sequences of Neanderthals and modern primates
    Nielsen-Marsh, Christina M.; Richards, Michael P.; Hauschka, Peter V.; Thomas-Oates, Jane E.; Trinkaus, Erik; Pettitt, Paul B.; Karavanic, Ivor; Poinar, Hendrik; Collins, Matthew J.
    Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (12), 4409-4413CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    We report here protein sequences of fossil hominids, from two Neanderthals dating to ≈ 75,000 years old from Shanidar Cave in Iraq. These sequences, the oldest reported fossil primate protein sequences, are of bone osteocalcin, which was extd. and sequenced by using MALDI-TOF/TOF mass spectrometry. Through a combination of direct sequencing and peptide mass mapping, we detd. that Neanderthals have an osteocalcin amino acid sequence that is identical to that of modern humans. We also report complete osteocalcin sequences for chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla gorilla) and a partial sequence for orangutan (Pongo pygmaeus), all of which are previously unreported. We found that the osteocalcin sequences of Neanderthals, modern human, chimpanzee, and orangutan are unusual among mammals in that the ninth amino acid is proline (Pro-9), whereas most species have hydroxyproline (Hyp-9). Posttranslational hydroxylation of Pro-9 in osteocalcin by prolyl-4-hydroxylase requires adequate concns. of vitamin C (L-ascorbic acid), mol. OZ, Fe2+, and 2-oxoglutarate, and also depends on enzyme recognition of the target proline substrate consensus sequence Leu-Gly-Ala-Pro-9-Ala-Pro-Tyr occurring in most mammals. In five species with Pro-9-Val-10, hydroxylation is blocked, whereas in gorilla there is a mixt. of Pro-9 and Hyp-9. We suggest that the absence of hydroxylation of Pro-9 in Pan, Pongo, and Homo may reflect response to a selective pressure related to a decline in vitamin C in the diet during omnivorous dietary adaptation, either independently or through the common ancestor of these species.
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  75. 75
    Buckley, M.; Anderung, C.; Penkman, K.; Raney, B. J.; Götherström, A.; Thomas-Oates, J.; Collins, M. J. Comparing the Survival of Osteocalcin and mtDNA in Archaeological Bone from Four European Sites. J. Archaeol. Sci. 2008, 35, 1756– 1764,  DOI: 10.1016/j.jas.2007.11.022
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  76. 76
    Buckley, M.; Collins, M.; Thomas-Oates, J. A Method of Isolating the Collagen (I) Alpha2 Chain Carboxytelopeptide for Species Identification in Bone Fragment. Anal. Biochem. 2008, 374, 325– 334,  DOI: 10.1016/j.ab.2007.12.002
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    76
    A method of isolating the collagen (I) α2 chain carboxytelopeptide for species identification in bone fragments
    Buckley, Michael; Collins, Matthew; Thomas-Oates, Jane
    Analytical Biochemistry (2008), 374 (2), 325-334CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    We present a novel method for the isolation and anal. of the bone collagen (I) α2 chain carboxytelopeptide as a species biomarker. Conventional methods for the anal. and sequencing of mixts. of proteins and peptides commonly involve using the protease trypsin to cleave proteins present in the sample. However, in the study of collagen, these methods result in very complex mixts. of peptides that are difficult to analyze and the acquired results are not reproducible. Here we use bacterial collagenase (from Clostridium histolyticum) for its ability to cleave the highly unusual Gly-Xaa-Yaa repeating sequence of collagen, where Xaa usually is Pro and Yaa often is Hyp. Followed by a simple isolation step using a reverse phase solid phase extn. cartridge, the α2 (I) chain carboxytelopeptide can be readily analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and the results can be used to distinguish between different species of origin.
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  77. 77
    Buckley, M. Zooarchaeology by Mass Spectrometry (ZooMS) Collagen Fingerprinting for the Species Identification of Archaeological Bone Fragments. In Zooarchaeology in Practice: Case Studies in Methodology and Interpretation in Archaeofaunal Analysis; Giovas, C. M., LeFebvre, M. J., Eds.; Springer International Publishing: Cham, 2018; pp 227– 247.  DOI: 10.1007/978-3-319-64763-0_12 .
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  78. 78
    Collins, M.; Buckley, M.; Grundy, H. H.; Thomas-Oates, J.; Wilson, J.; van Doorn, N. ZooMS: The Collagen Barcode and Fingerprints. Spectrosc. Eur. 2010, 22, 6– 10
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    78
    ZooMS: the collagen barcode and fingerprints
    Collins, Matthew; Buckley, Mike; Grundy, Helen H.; Thomas-Oates, Jane; Wilson, Julie; van Doorn, Nienke
    Spectroscopy Europe (2010), 22 (2), 6-8, 10CODEN: SPEUEF; ISSN:0966-0941. (John Wiley & Sons Ltd.)
    This year, the International Barcode of Life initiative (IBoL) plans to begin an ambitious program to barcode the DNA of more than five million specimens representing at least 500,000 species in five years. In line with this, the use of peptide fingerprinting, an approach termed ZooMS, short for Zooarchaeol. by Mass Spectrometry is described. Notably, the fact that collagen is selectively preserved in old bone (and teeth, ivory and antler) means that it can be used as a mol. barcode long after DNA barcodes have been damaged, fragmented and have 'melted' away. Generally, the particular characteristics of mass spectrometry have the potential to revolutionize the identification of animals' tissues, even from tiny fragments, far back into time and across the vertebrate kingdom. It is argued that just as DNA can be considered a barcode of life, collagen has the potential to be the barcode for the communities of the dead.
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  79. 79
    Janzen, A.; Richter, K. K.; Mwebi, O.; Brown, S.; Onduso, V.; Gatwiri, F.; Ndiema, E.; Katongo, M.; Goldstein, S. T.; Douka, K. Distinguishing African Bovids Using Zooarchaeology by Mass Spectrometry (ZooMS): New Peptide Markers and Insights into Iron Age Economies in Zambia. PLoS One 2021, 16, e0251061  DOI: 10.1371/journal.pone.0251061
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    79
    Distinguishing African bovids using Zooarchaeology by Mass Spectrometry (ZooMS): New peptide markers and insights into Iron Age economies in Zambia
    Janzen, Anneke; Richter, Kristine Korzow; Mwebi, Ogeto; Brown, Samantha; Onduso, Veronicah; Gatwiri, Filia; Ndiema, Emmanuel; Katongo, Maggie; Goldstein, Steven T.; Douka, Katerina; Boivin, Nicole
    PLoS One (2021), 16 (5), e0251061CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Assessing past foodways, subsistence strategies, and environments depends on the accurate identification of animals in the archaeol. record. The high rates of fragmentation and often poor preservation of animal bones at many archaeol. sites across sub-Saharan Africa have rendered archaeofaunal specimens unidentifiable beyond broad categories, such as "large mammal" or "medium bovid". Identification of archaeofaunal specimens through Zooarchaeol. by Mass Spectrometry (ZooMS), or peptide mass fingerprinting of bone collagen, offers an avenue for identification of morphol. ambiguous or unidentifiable bone fragments from such assemblages. However, application of ZooMS anal. has been hindered by a lack of complete ref. peptide markers for African taxa, particularly bovids. Here we present the complete set of confirmed ZooMS peptide markers for members of all African bovid tribes. We also identify two novel peptide markers that can be used to further distinguish between bovid groups. We demonstrate that nearly all African bovid subfamilies are distinguishable using ZooMS methods, and some differences exist between tribes or sub-tribes, as is the case for Bovina (cattle) vs. Bubalina (African buffalo) within the subfamily Bovinae. We use ZooMS anal. to identify specimens from extremely fragmented faunal assemblages from six Late Holocene archaeol. sites in Zambia. ZooMS-based identifications reveal greater taxonomic richness than analyses based solely on morphol., and these new identifications illuminate Iron Age subsistence economies c. 2200-500 cal BP. While the Iron Age in Zambia is assocd. with the transition from hunting and foraging to the development of farming and herding, our results demonstrate the continued reliance on wild bovids among Iron Age communities in central and southwestern Zambia Iron Age and herding focused primarily on cattle. We also outline further potential applications of ZooMS in African archaeol.
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  80. 80
    Buckley, M.; Fraser, S.; Herman, J.; Melton, N. D.; Mulville, J.; Pálsdóttir, A. H. Species Identification of Archaeological Marine Mammals Using Collagen Fingerprinting. J. Archaeol. Sci. 2014, 41, 631– 641,  DOI: 10.1016/j.jas.2013.08.021
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    80
    Species identification of archaeological marine mammals using collagen fingerprinting
    Buckley, M.; Fraser, S.; Herman, J.; Melton, N. D.; Mulville, J.; Palsdottir, A. H.
    Journal of Archaeological Science (2014), 41 (), 631-641CODEN: JASCDU; ISSN:1095-9238. (Elsevier Inc.)
    Throughout human history, coastal and marine resources have been a vital part of human subsistence. As a result archaeol. faunal assemblages from coastal sites often contain large quantities of skeletal remains indicative of human interaction with marine mammals. However, these are often hard to identify due to a unique combination of factors regarding the procurement, utilization, morphol. and phys. characteristics of marine mammal bones. These factors often result in a large no. of archaeol. cetacean and pinniped specimens fragmented beyond visual recognition, being labeled 'whale' or 'marine mammal'. In this paper we report the development of a Zooarchaeol. by Mass Spectrometry (ZooMS) method of collagen fingerprinting, for efficient and low cost discrimination of a wide range of marine mammal species including cetaceans and pinnipeds. We apply the technique to more than fifty archaeol. specimens from seven different North Atlantic sites ranging from the Mesolithic until the Early Modern period.
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  81. 81
    Harvey, V. L.; Daugnora, L.; Buckley, M. Species Identification of Ancient Lithuanian Fish Remains Using Collagen Fingerprinting. J. Archaeol. Sci. 2018, 98, 102– 111,  DOI: 10.1016/j.jas.2018.07.006
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  82. 82
    Eda, M.; Morimoto, M.; Mizuta, T.; Inoué, T. ZooMS for Birds: Discrimination of Japanese Archaeological Chickens and Indigenous Pheasants Using Collagen Peptide Fingerprinting. J. Archaeol. Sci. Rep. 2020, 34, 102635,  DOI: 10.1016/j.jasrep.2020.102635
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  83. 83
    Harvey, V. L.; LeFebvre, M. J.; deFrance, S. D.; Toftgaard, C.; Drosou, K.; Kitchener, A. C.; Buckley, M. Preserved Collagen Reveals Species Identity in Archaeological Marine Turtle Bones from Caribbean and Florida Sites. R. Soc. Open Sci. 2019, 6, 191137,  DOI: 10.1098/rsos.191137
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    83
    Preserved collagen reveals species identity in archaeological marine turtle bones from Caribbean and Florida sites
    Harvey, Virginia L.; LeFebvre, Michelle J.; deFrance, Susan D.; Toftgaard, Casper; Drosou, Konstantina; Kitchener, Andrew C.; Buckley, Michael
    Royal Society Open Science (2019), 6 (10), 191137CODEN: RSOSAV; ISSN:2054-5703. (Royal Society)
    Advancements in mol. science are continually improving our knowledge of marine turtle biol. and evolution. However, there are still considerable gaps in our understanding, such as past marine turtle distributions, which can benefit from advanced zooarchaeol. analyses. Here, we apply collagen fingerprinting to 130 archaeol. marine turtle bone samples up to approx. 2500 years old from the Caribbean and Florida's Gulf Coast for faunal identification, finding the vast majority of samples (88%) to contain preserved collagen despite deposition in the tropics. All samples can be identified to species-level with the exception of the Kemp's ridley (Lepidochelys kempii) and olive ridley (L. olivacea) turtles, which can be sepd. to genus level, having diverged from one another only approx. 5 Ma. Addnl., we identify a single homologous peptide that allows the sepn. of archaeol. green turtle samples, Chelonia spp., into two distinct groups, which potentially signifies a difference in genetic stock. The majority of the archaeol. samples are identified as green turtle (Chelonia spp.; 63%), with hawksbill (Eretmochelys imbricata; 17%) and ridley turtles (Lepidochelys spp.; 3%) making up smaller proportions of the assemblage. There were no mol. identifications of the loggerhead turtle (Caretta caretta) in the assemblage despite 9% of the samples being morphol. identified as such, highlighting the difficulties in relying on morphol. identifications alone in archaeol. remains. Finally, we present the first marine turtle mol. phylogeny using collagen (I) amino acid sequences and find our analyses match recent phylogenies based on nuclear and mitochondrial DNA. Our results highlight the advantage of using collagen fingerprinting to supplement morphol. analyses of turtle bones and support the usefulness of this technique for assessing their past distributions across the Caribbean and Florida's Gulf Coast, esp. in these tropical environments where DNA preservation may be poor.
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  84. 84
    Solazzo, C.; Wadsley, M.; Dyer, J. M.; Clerens, S.; Collins, M. J.; Plowman, J. Characterisation of Novel α-Keratin Peptide Markers for Species Identification in Keratinous Tissues Using Mass Spectrometry. Rapid Commun. Mass Spectrom. 2013, 27, 2685– 2698,  DOI: 10.1002/rcm.6730
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    84
    Characterisation of novel α-keratin peptide markers for species identification in keratinous tissues using mass spectrometry
    Solazzo, Caroline; Wadsley, Marc; Dyer, Jolon M.; Clerens, Stefan; Collins, Matthew J.; Plowman, Jeffrey
    Rapid Communications in Mass Spectrometry (2013), 27 (23), 2685-2698CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    RATIONALE : In ancient and/or damaged artifacts contg. keratinous materials, the species of origin of the materials can be difficult to identify through visual examn.; therefore, a minimally destructive methodol. for species identification is required. While hair fibers from some species have seen substantial characterization, others such as horn or baleen have received little or no attention, or lack protein sequences allowing formal identification using proteomics techniques. METHODS : We used the PMF method (Peptide Mass Fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)) to catalog and identify diagnostic peptide markers up to the genus level. Sequences were checked using nanoflow liq. chromatog./electrospray ionization tandem mass spectrometry (nanoLC/ESI-MS/MS) and unidentified peptides were searched against a theor. database generated by substituting amino acids in keratin sequences. RESULTS : Specific peptides were identified by m/z and sequences characterized whenever possible for a range of species belonging to Bovidae and Camelidae, and for tissues such as baleen and horn. The theor. database allowed an increase in the no. of peptides of up to 10% in species with little genetic information. CONCLUSIONS : A proteomics approach can successfully identify specific markers for the identification of materials to the genus level, and should be considered when identification by other means is not possible. Identification by PMF is fast, reliable and inexpensive. Copyright © 2013 John Wiley & Sons, Ltd.
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  85. 85
    Hollemeyer, K.; Altmeyer, W.; Heinzle, E. Identification and Quantification of Feathers, Down, and Hair of Avian and Mammalian Origin Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Anal. Chem. 2002, 74, 5960– 5968,  DOI: 10.1021/ac020347f
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    85
    Identification and quantification of feathers, down, and hair of avian and mammalian origin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
    Hollemeyer, Klaus; Altmeyer, Wolfgang; Heinzle, Elmar
    Analytical Chemistry (2002), 74 (23), 5960-5968CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    We describe a fast method for the identification of the origin of native and chem. processed feathers, down, and hair and for distinguishing closely related species using enzyme digestion and MALDI-TOF mass spectrometry. Addnl. we present two methods for the quantification of different identified bird and mammalian samples, resp., in binary species mixts. Without any prior cleaning or isolation of single proteins, enzymical digests of feathers, down, and hair are performed. Fragments generated are analyzed by MALDI-TOF mass spectrometry, and peak groups of different selectivity are established for every animal species. For the identification of individual animal species, only unique species-specific peaks are accepted while for species classification of tinted or bleached hair, the group of semispecific peaks (SEMPs) is used. Samples from native animal species show a higher coincidence of digest peak masses of SEMPs than far-related species, indicating a phylogenetical relationship of the investigated structure proteins. Quantification of mixed binary avian samples is performed by identification of preweighed single feathers and down, followed by calcn. of the gravimetric mass fractions. The compn. of mixed binary mammalian samples is calcd. from correlation of the quotient of the relative peak intensities or areas of these species and the quotient of corresponding gravimetric mass fractions. High accuracy is achieved by both quantification methods. The fast methods are well suited for industrial quality control for example.
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  86. 86
    Presslee, S.; Wilson, J.; Woolley, J.; Best, J.; Russell, D.; Radini, A.; Fischer, R.; Kessler, B.; Boano, R.; Collins, M. The Identification of Archaeological Eggshell Using Peptide Markers. Sci. Technol. Archaeol. 2017, 3, 89– 99,  DOI: 10.1080/20548923.2018.1424300
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  87. 87
    Stewart, J. R. M.; Allen, R. B.; Jones, A. K. G.; Penkman, K. E. H.; Collins, M. J. ZooMS: Making Eggshell Visible in the Archaeological Record. J. Archaeol. Sci. 2013, 40, 1797– 1804,  DOI: 10.1016/j.jas.2012.11.007
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    87
    ZooMS: making eggshell visible in the archaeological record
    Stewart, John R. M.; Allen, Richard B.; Jones, Andrew K. G.; Penkman, Kirsty E. H.; Collins, Matthew J.
    Journal of Archaeological Science (2013), 40 (4), 1797-1804CODEN: JASCDU; ISSN:1095-9238. (Elsevier Inc.)
    Avian eggshell is a common component of many archaeol. deposits, but its archaeol. potential remains largely unexplored. The most obvious reasons are two-fold. Firstly, despite its abundance on many sites, eggshell is often overlooked during excavation. Even when it is recovered, small fragmented remains are difficult to identify taxonomically. Here, we introduce a minimally destructive qual. anal. technique for taxonomic identification of eggshell fragments based on highly sensitive mass spectrometry and peptide mass fingerprinting (ZooMS), and illustrate its application to eggshell recovered from the Viking Age urban site at Hungate, York. We adopt a more extreme version of the method of bleach treating used to prep. ancient eggshell for DNA anal., followed by conventional peptide mass fingerprinting using MALDI-ToF mass spectrometry. The development of this technique will allow future research to make better use of eggshell fragments recovered from archaeol. sites.
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  88. 88
    Sakalauskaite, J.; Marin, F.; Pergolizzi, B.; Demarchi, B. Shell Palaeoproteomics: First Application of Peptide Mass Fingerprinting for the Rapid Identification of Mollusc Shells in Archaeology. J. Proteomics 2020, 227, 103920,  DOI: 10.1016/j.jprot.2020.103920
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    88
    Shell palaeoproteomics: First application of peptide mass fingerprinting for the rapid identification of mollusc shells in archaeology
    Sakalauskaite, Jorune; Marin, Frederic; Pergolizzi, Barbara; Demarchi, Beatrice
    Journal of Proteomics (2020), 227 (), 103920CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)
    Molluscs were one of the most widely-used natural resources in the past, and their shells are abundant among archaeol. findings. However, our knowledge of the variety of shells that were circulating in prehistoric times (and thus their socio-economic and cultural value) is scarce due to the difficulty of achieving taxonomic detn. of fragmented and/or worked remains. This study aims to obtain mol. barcodes based on peptide mass fingerprints (PMFs) of intracryst. proteins, in order to obtain shell identification. Palaeoproteomic applications on shells are challenging, due to low concn. of molluscan proteins and an incomplete understanding of their sequences. We explore different approaches for protein extn. from small-size samples (<20 mg), followed by MALDI-TOF-MS anal. The SP3 (single-pot, solid-phase) sample prepn. method was found to be the most successful in retrieving the intracryst. protein fraction from seven molluscan shell taxa, which belong to different phylogenetic groups, possess distinct microstructures and are relevant for archaeol. Furthermore, all the shells analyzed, including a 7000-yr-old specimen of the freshwater bivalve Pseudunio, yielded good-quality distinctive spectra, demonstrating that PMFs can be used for shell taxon detn. Our work suggests good potential for large-scale screening of archaeol. molluscan remains. We characterize for the first time the peptide mass fingerprints of the intracryst. shell protein fraction isolated from different molluscan taxa. We demonstrate that these proteins yield distinctive PMFs, even for shells that are phylogenetically related and/or that display similar microstructures. Furthermore, we extend the range of sample prepn. approaches for "shellomics" by testing the SP3 method, which proved to be well-suited to shell protein extn. from small-size and protein-poor samples. This work thus lays the foundations for future large-scale applications for the identification of mollusc shells and other invertebrate remains from the archaeol. and palaeontol. records.
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  89. 89
    Tokarski, C.; Martin, E.; Rolando, C.; Cren-Olivé, C. Identification of Proteins in Renaissance Paintings by Proteomics. Anal. Chem. 2006, 78, 1494– 1502,  DOI: 10.1021/ac051181w
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    89
    Identification of Proteins in Renaissance Paintings by Proteomics
    Tokarski, Caroline; Martin, Elisabeth; Rolando, Christian; Cren-Olive, Cecile
    Analytical Chemistry (2006), 78 (5), 1494-1502CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The presented work proposes a new methodol. based on proteomics techniques to identify proteins in old art paintings. The main challenging tasks of this work were (i) to find appropriate conditions for extg. proteins from the binding media without protein hydrolysis to amino acids and (ii) to develop anal. methods adapted to the small sample quantity available. Starting from microsamples of painting models (ovalbumin-based, which is the major egg white protein, and egg-based paintings), multiple extn. solns. (HCl, HCOOH, NH3, NaOH) and conditions (ultrasonic bath, mortar and pestle, grinding resin) were evaluated. The best results were obtained using a com. kit including a synthetic resin, mortar and pestle to grind the sample in an aq. soln. acidified with trifluoroacetic acid at 1% with addnl. multiple steps of ultrasonic baths. The resulting supernatant was analyzed by MALDI-TOF in linear mode to verify the efficiency of the extn. soln. An enzymic hydrolysis step was also performed for protein identification; the peptide mixt. was analyzed by nanoLC/nanoESI/Q-q-TOF MS/MS with an adapted chromatog. run for the low sample quantity. Finally, the developed methodol. was successfully applied to Renaissance art painting microsamples of ∼10 μg from Benedetto Bonfigli's triptych, The Virgin and Child, St. John the Baptist, St. Sebastian (XVth century), and Niccolo di Pietro Gerini's painting, The Virgin and Child (XIVth century), identifying, for the first time and without ambiguity, the presence of whole egg proteins (egg yolk and egg white) in a painting binder.
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  90. 90
    Zubarev, R. A.; Makarov, A. Orbitrap Mass Spectrometry. Anal. Chem. 2013, 85, 5288– 5296,  DOI: 10.1021/ac4001223
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    90
    Orbitrap Mass Spectrometry
    Zubarev, Roman A.; Makarov, Alexander
    Analytical Chemistry (Washington, DC, United States) (2013), 85 (11), 5288-5296CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A review. Orbitrap is the newest addn. to the family of high-resoln. mass spectrometry analyzers. With its revolutionarily new, miniature design, Orbitrap combines high speed with excellent quantification properties, ranking favorably in many anal. applications.
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  91. 91
    Cappellini, E.; Jensen, L. J.; Szklarczyk, D.; Ginolhac, A.; da Fonseca, R. A. R.; Stafford, T. W., Jr.; Holen, S. R.; Collins, M. J.; Orlando, L.; Willerslev, E. Proteomic Analysis of a Pleistocene Mammoth Femur Reveals More than One Hundred Ancient Bone Proteins. J. Proteome Res. 2012, 11, 917– 926,  DOI: 10.1021/pr200721u
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    91
    Proteomic Analysis of a Pleistocene Mammoth Femur Reveals More than One Hundred Ancient Bone Proteins
    Cappellini, Enrico; Jensen, Lars J.; Szklarczyk, Damian; Ginolhac, Aurelien; da Fonseca, Rute A. R.; Stafford, Thomas W.; Holen, Steven R.; Collins, Matthew J.; Orlando, Ludovic; Willerslev, Eske; Gilbert, M. Thomas P.; Olsen, Jesper V.
    Journal of Proteome Research (2012), 11 (2), 917-926CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    We used high-sensitivity, high-resoln. tandem mass spectrometry to shotgun sequence ancient protein remains extd. from a 43 000 yr old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance extracellular matrix and plasma proteins, were confidently identified by solid mol. evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African (Loxodonta africana) and Indian (Elephas maximus) elephants. Strong evidence was obsd. of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth (Mammuthus columbi) samples from temperate latitudes, extending the potential of the approach described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future mol. phylogenetic inference and physiol. studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing integration of different high-throughput technologies for identification of ancient biomols., unleashing the field of paleoproteomics.
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  92. 92
    Warinner, C.; Rodrigues, J. F. M.; Vyas, R.; Trachsel, C.; Shved, N.; Grossmann, J.; Radini, A.; Hancock, Y.; Tito, R. Y.; Fiddyment, S. Pathogens and Host Immunity in the Ancient Human Oral Cavity. Nat. Genet. 2014, 46, 336– 344,  DOI: 10.1038/ng.2906
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    92
    Pathogens and host immunity in the ancient human oral cavity
    Warinner, Christina; Rodrigues, Joao F. Matias; Vyas, Rounak; Trachsel, Christian; Shved, Natallia; Grossmann, Jonas; Radini, Anita; Hancock, Y.; Tito, Raul Y.; Fiddyment, Sarah; Speller, Camilla; Hendy, Jessica; Charlton, Sophy; Luder, Hans Ulrich; Salazar-Garcia, Domingo C.; Eppler, Elisabeth; Seiler, Roger; Hansen, Lars H.; Castruita, Jose Alfredo Samaniego; Barkow-Oesterreicher, Simon; Teoh, Kai Yik; Kelstrup, Christian D.; Olsen, Jesper V.; Nanni, Paolo; Kawai, Toshihisa; Willerslev, Eske; von Mering, Christian; Lewis, Cecil M., Jr.; Collins, Matthew J.; Gilbert, M. Thomas P.; Ruehli, Frank; Cappellini, Enrico
    Nature Genetics (2014), 46 (4), 336-344CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)
    Calcified dental plaque (dental calculus) preserves for millennia and entraps biomols. from all domains of life and viruses. We report the first, to our knowledge, high-resoln. taxonomic and protein functional characterization of the ancient oral microbiome and demonstrate that the oral cavity has long served as a reservoir for bacteria implicated in both local and systemic disease. We characterize (i) the ancient oral microbiome in a diseased state, (ii) 40 opportunistic pathogens, (iii) ancient human-assocd. putative antibiotic resistance genes, (iv) a genome reconstruction of the periodontal pathogen Tannerella forsythia, (v) 239 bacterial and 43 human proteins, allowing confirmation of a long-term assocn. between host immune factors, 'red complex' pathogens and periodontal disease, and (vi) DNA sequences matching dietary sources. Directly datable and nearly ubiquitous, dental calculus permits the simultaneous investigation of pathogen activity, host immunity and diet, thereby extending direct investigation of common diseases into the human evolutionary past.
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  93. 93
    Buckley, M. Ancient Collagen Reveals Evolutionary History of the Endemic South American “ungulates.. Proceedings of the Royal Society B: Biological Sciences 2015, 282, 20142671,  DOI: 10.1098/rspb.2014.2671
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  94. 94
    Stewart, N. A.; Gerlach, R. F.; Gowland, R. L.; Gron, K. J.; Montgomery, J. Sex Determination of Human Remains from Peptides in Tooth Enamel. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 13649– 13654,  DOI: 10.1073/pnas.1714926115
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    94
    Sex determination of human remains from peptides in tooth enamel
    Stewart, Nicolas Andre; Gerlach, Raquel Fernanda; Gowland, Rebecca L.; Gron, Kurt J.; Montgomery, Janet
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (52), 13649-13654CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The assignment of biol. sex to archaeol. human skeletons is a fundamental requirement for the reconstruction of the human past. It is conventionally and routinely performed on adults using metric anal. and morphol. traits arising from postpubertal sexual dimorphism. A max. accuracy of ∼95% is possible if both the cranium and os coxae are present and intact, but this is seldom achievable for all skeletons. Furthermore, for infants and juveniles, there are no reliable morphol. methods for sex detn. without resorting to DNA anal., which requires good DNA survival and is time-consuming. MS/MS. Consequently, sex detn. of juvenile remains is rarely undertaken, and a dependable and expedient method that can correctly assign biol. sex to human remains of any age is highly desirable. Here the authors present a method for sex detn. of human remains by means of a minimally destructive surface acid etching of tooth enamel and subsequent identification of sex chromosome-linked isoforms of amelogenin, an enamel-forming protein, by nanoflow liq. chromatog. mass spectrometry. Tooth enamel is the hardest tissue in the human body and survives burial exceptionally well, even when the rest of the skeleton or DNA in the org. fraction has decayed. The method can reliably det. the biol. sex of humans of any age using a body tissue that is difficult to cross-contaminate and is most likely to survive. The application of this method will make sex detn. of adults and, for the first time, juveniles a reliable and routine activity in future bioarcheol. and medico-legal science contexts.
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  95. 95
    Shevchenko, A.; Yang, Y.; Knaust, A.; Thomas, H.; Jiang, H.; Lu, E.; Wang, C.; Shevchenko, A. Proteomics Identifies the Composition and Manufacturing Recipe of the 2500-Year Old Sourdough Bread from Subeixi Cemetery in China. J. Proteomics 2013,  DOI: 10.1016/j.jprot.2013.11.016
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  96. 96
    Bona, A.; Papai, Z.; Maasz, G.; Toth, G. A.; Jambor, E.; Schmidt, J.; Toth, C.; Farkas, C.; Mark, L. Mass Spectrometric Identification of Ancient Proteins as Potential Molecular Biomarkers for a 2000-Year-Old Osteogenic Sarcoma. PLoS One 2014, 9, e87215  DOI: 10.1371/journal.pone.0087215
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    96
    Mass spectrometric identification of ancient proteins as potential molecular biomarkers for a 2000-year-old osteogenic sarcoma
    Bona, Agnes; Papai, Zoltan; Maasz, Gabor; Toth, Gabor A.; Jambor, Eva; Schmidt, Janos; Toth, Csaba; Farkas, Csilla; Mark, Laszlo
    PLoS One (2014), 9 (1), e87215/1-e87215/9, 9 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Osteosarcoma is the most common primary malignant tumor of bone usually occurring in young adolescent and children. This disease has a poor prognosis, because of the metastases in the period of tumor progression, which are usually developed previous to the clin. diagnosis. In this paper, a 2000-yr-old ancient bone remain with osteogenic sarcoma was analyzed searching for tumor biomarkers which are closely related to this disease. After a specific extn. SDS-PAGE gel electrophoresis followed by tryptic digestion was performed. After the digestion the samples were measured using MALDI TOF/TOF MS. Healthy bone samples from same archaeol. site were used as control samples. Our results show that in the pathol. skeletal remain several well known tumor biomarkers are detected such as annexin A10, BCL-2-like protein, calgizzarin, rho GTPase-activating protein 7, HSP beta-6 protein, transferrin and vimentin compared to the control samples. The identified protein biomarkers can be useful in the discovery of malignant bone lesions such as osteosarcoma in the very early stage of the disease from paleoanthropol. remains.
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  97. 97
    Lluveras-Tenorio, A.; Vinciguerra, R.; Galano, E.; Blaensdorf, C.; Emmerling, E.; Perla Colombini, M.; Birolo, L.; Bonaduce, I. GC/MS and Proteomics to Unravel the Painting History of the Lost Giant Buddhas of Ba̅miya̅n (Afghanistan). PLoS One 2017, 12, e0172990  DOI: 10.1371/journal.pone.0172990
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    97
    GC/MS and proteomics to unravel the painting history of the lost Giant Buddhas of Bamiyan (Afghanistan)
    Lluveras-Tenorio, Anna; Vinciguerra, Roberto; Galano, Eugenio; Blaensdorf, Catharina; Emmerling, Erwin; Colombini, Maria Perla; Birolo, Leila; Bonaduce, Ilaria
    PLoS One (2017), 12 (4), e0172990/1-e0172990/18CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    A chem. investigation of the org. paint binders of the Giant Buddhas of Bamiyan was performed using an anal. approach based on mass spectrometry, combining traditional gas chromatog./mass spectrometry protocols with advanced proteomics methodologies. The research was carried out on a selection of rescued fragments. The data revealed the use of egg proteins as the paint binders of the original layers, in accordance with the traditional use of this proteinaceous medium in antiquity, spanning from the Mediterranean basin to the Far East, and already in the Bronze Age. Egg tempera was thus known to artists of the region in the first centuries AD, probably also due to the position of the Bamiyan valley, which was connected to the Silk Road. Milk was found in the first historical overpaintings. A new proteomics approach was used, which was able to identify the source of the milk proteins present in the restoration layers, despite their age and degrdn. In particular cow's and goat's milk were both found, in agreement with the documented presence of rich pastures in the Bamiyan valley when the historical restorations were carried out. Investigating the materials of the Giant Buddhas not only enabled us to obtain isolated data on these invaluable works of art, which are now lost, but contributes to understanding the big "puzzle" of our past and the development of our culture, by implementing and supporting written sources, stylistic and anthropol. studies with mol. data.
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  98. 98
    Fremout, W.; Kuckova, S.; Crhova, M.; Sanyova, J.; Saverwyns, S.; Hynek, R.; Kodicek, M.; Vandenabeele, P.; Moens, L. Classification of Protein Binders in Artist’s Paints by Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry: An Evaluation of Principal Component Analysis (PCA) and Soft Independent Modelling of Class Analogy (SIMCA). Rapid Commun. Mass Spectrom. 2011, 25, 1631– 1640,  DOI: 10.1002/rcm.5027
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    98
    Classification of protein binders in artist's paints by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: an evaluation of principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA)
    Fremout, Wim; Kuckova, Stepanka; Crhova, Michaela; Sanyova, Jana; Saverwyns, Steven; Hynek, Radovan; Kodicek, Milan; Vandenabeele, Peter; Moens, Luc
    Rapid Communications in Mass Spectrometry (2011), 25 (11), 1631-1640CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Proteomics techniques are increasingly applied for the identification of protein binders in historical paints. The complex nature of paint samples, with different kinds of pigments mixed into, and degrdn. by long term exposure to light, humidity and temp. variations, requires solid anal. and interpretation methods. In this study matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of tryptic-digested paint replicas are subjected to principal component anal. (PCA) and soft independent modeling of class analogy (SIMCA) in order to distinguish proteinaceous binders based on animal glues, egg white, egg yolk and milk casein from each other. The most meaningful peptide peaks for a given protein class will be detd., and if possible, annotated with their corresponding amino acid sequence. The methodol. was subsequently applied on egg temperas, as well as on animal glues from different species. In the latter small differences in the MALDI-TOF mass spectra can allow the detn. of a mammal or sturgeon origin of the glue. Finally, paint samples from the 16th century altarpiece of St Margaret of Antioch (Mlynica, Slovakia) were analyzed. Several expected peaks are either present in lower abundance or completely missing in these natural aged paints, due to degrdn. of the paints. In spite of this mammalian glue was identified in the St Margaret samples. Copyright © 2011 John Wiley & Sons, Ltd.
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  99. 99
    Hendy, J.; Colonese, A. C.; Franz, I.; Fernandes, R.; Fischer, R.; Orton, D.; Lucquin, A.; Spindler, L.; Anvari, J.; Stroud, E. Ancient Proteins from Ceramic Vessels at Çatalhöyük West Reveal the Hidden Cuisine of Early Farmers. Nat. Commun. 2018, 9, 4064,  DOI: 10.1038/s41467-018-06335-6
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    99
    Ancient proteins from ceramic vessels at Catalhoyuk West reveal the hidden cuisine of early farmers
    Hendy Jessica; Fernandes Ricardo; Boivin Nicole; Hendy Jessica; Colonese Andre C; Orton David; Lucquin Alexandre; Spindler Luke; Speller Camilla; Collins Matthew J; Craig Oliver E; Franz Ingmar; Fernandes Ricardo; Stroud Elizabeth; Fischer Roman; Spindler Luke; Anvari Jana; Rosenstock Eva; Biehl Peter F; Speller Camilla; Mackie Meaghan; Collins Matthew J; Mackie Meaghan; Jersie-Christensen Rosa R; Olsen Jesper V
    Nature communications (2018), 9 (1), 4064 ISSN:.
    The analysis of lipids (fats, oils and waxes) absorbed within archaeological pottery has revolutionized the study of past diets and culinary practices. However, this technique can lack taxonomic and tissue specificity and is often unable to disentangle signatures resulting from the mixing of different food products. Here, we extract ancient proteins from ceramic vessels from the West Mound of the key early farming site of Catalhoyuk in Anatolia, revealing that this community processed mixes of cereals, pulses, dairy and meat products, and that particular vessels may have been reserved for specialized foods (e.g., cow milk and milk whey). Moreover, we demonstrate that dietary proteins can persist on archaeological artefacts for at least 8000 years, and that this approach can reveal past culinary practices with more taxonomic and tissue-specific clarity than has been possible with previous biomolecular techniques.
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  100. 100
    Gorski, J. P. Biomineralization of Bone: A Fresh View of the Roles of Non-Collagenous Proteins. Front. Biosci. 2011, 16, 2598– 2621,  DOI: 10.2741/3875
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    100
    Biomineralization of bone: a fresh view of the roles of non-collagenous proteins
    Gorski, Jeffrey Paul
    Frontiers in Bioscience, Landmark Edition (2011), 16 (7), 2598-2621CODEN: FRBIF6; ISSN:1093-4715. (Frontiers in Bioscience)
    A review. The impact of genetics has dramatically affected our understanding of the functions of non-collagenous proteins. Specifically, mutations and knockouts have defined their cellular spectrum of actions. However, the biochem. mechanisms mediated by non-collagenous proteins in biomineralization remain elusive. It is likely that this understanding will require more focused functional testing at the protein, cell, and tissue level. Although initially viewed as rather redundant and static acidic calcium binding proteins, it is now clear that non-collagenous proteins in mineralizing tissues represent diverse entities capable of forming multiple protein-protein interactions which act in pos. and neg. ways to regulate the process of bone mineralization. Several new examples from the author's lab. are provided which illustrate this theme including an apparent activating effect of hydroxyapatite crystals on metalloproteinases. This review emphasizes the view that secreted non-collagenous proteins in mineralizing bone actively participate in the mineralization process and ultimately control where and how much mineral crystal is deposited, as well as detg. the quality and biomech. properties of the mineralized matrix produced.
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  101. 101
    Wilkin, S.; Ventresca Miller, A.; Taylor, W. T. T.; Miller, B. K.; Hagan, R. W.; Bleasdale, M.; Scott, A.; Gankhuyg, S.; Ramsøe, A.; Trachsel, C. Dairy Pastoralism Sustained Eastern Eurasian Steppe Populations for 5000 Years. Nat. Ecol. Evol. 2020, 4, 346– 355,  DOI: 10.1038/s41559-020-1120-y
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    101
    Dairy pastoralism sustained eastern Eurasian steppe populations for 5,000 years
    Wilkin Shevan; Ventresca Miller Alicia; Taylor William T T; Miller Bryan K; Bleasdale Madeleine; Horton Mark; Boivin Nicole; Hendy Jessica; Ventresca Miller Alicia; Taylor William T T; Miller Bryan K; Hagan Richard W; Scott Ashley; Stockhammer Philipp W; Warinner Christina; Hendy Jessica; Gankhuyg Sumiya; Myagmar Erdene; Ramsoe Abigail; Hendy Jessica; Ramsoe Abigail; Uliziibayar S; Trachsel Christian; Nanni Paolo; Grossmann Jonas; Orlando Ludovic; Orlando Ludovic; Horton Mark; Stockhammer Philipp W; Boivin Nicole; Boivin Nicole; Boivin Nicole; Warinner Christina; Warinner Christina
    Nature ecology & evolution (2020), 4 (3), 346-355 ISSN:.
    Dairy pastoralism is integral to contemporary and past lifeways on the eastern Eurasian steppe, facilitating survival in agriculturally challenging environments. While previous research has indicated that ruminant dairy pastoralism was practiced in the region by circa 1300 BC, the origin, extent and diversity of this custom remain poorly understood. Here, we analyse ancient proteins from human dental calculus recovered from geographically diverse locations across Mongolia and spanning 5,000 years. We present the earliest evidence for dairy consumption on the eastern Eurasian steppe by circa 3000 BC and the later emergence of horse milking at circa 1200 BC, concurrent with the first evidence for horse riding. We argue that ruminant dairying contributed to the demographic success of Bronze Age Mongolian populations and that the origins of traditional horse dairy products in eastern Eurasia are closely tied to the regional emergence of mounted herding societies during the late second millennium BC.
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  102. 102
    Scott, A.; Power, R. C.; Altmann-Wendling, V.; Artzy, M.; Martin, M. A. S.; Eisenmann, S.; Hagan, R.; Salazar-García, D. C.; Salmon, Y.; Yegorov, D. Exotic Foods Reveal Contact between South Asia and the Near East during the Second Millennium BCE. Proc. Natl. Acad. Sci. U. S. A. 2021, 118, e2014956117  DOI: 10.1073/pnas.2014956117
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    102
    Exotic foods reveal contact between South Asia and the Near East during the second millennium BCE
    Scott, Ashley; Power, Robert C.; Altmann-Wendling, Victoria; Artzy, Michal; Martin, Mario A. S.; Eisenmann, Stefanie; Hagan, Richard; Salazar-Garcia, Domingo C.; Salmon, Yossi; Yegorov, Dmitry; Milevski, Ianir; Finkelstein, Israel; Stockhammer, Philipp W.; Warinner, Christina
    Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (2), e2014956117CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Although the key role of long-distance trade in the transformation of cuisines worldwide has been well-documented since at least the Roman era, the prehistory of the Eurasian food trade is less visible. In order to shed light on the transformation of Eastern Mediterranean cuisines during the Bronze Age and Early Iron Age, we analyzed microremains and proteins preserved in the dental calculus of individuals who lived during the second millennium BCE in the Southern Levant. Our results provide clear evidence for the consumption of expected staple foods, such as cereals (Triticeae), sesame (Sesamum), and dates (Phoenix). We addnl. report evidence for the consumption of soybean (Glycine), probable banana (Musa), and turmeric (Curcuma), which pushes back the earliest evidence of these foods in the Mediterranean by centuries (turmeric) or even millennia (soybean). We find that, from the early second millennium onwards, at least some people in the Eastern Mediterranean had access to food from distant locations, including South Asia, and such goods were likely consumed as oils, dried fruits, and spices. These insights force us to rethink the complexity and intensity of Indo-Mediterranean trade during the Bronze Age as well as the degree of globalization in early Eastern Mediterranean cuisine.
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  103. 103
    Drooker, P. B. Introduction. In Perishable material culture in the Northeast; Drooker, P. B., Ed.; New York State Museum: Albany, NY, 2004; pp 1– 18.
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  104. 104
    Solazzo, C.; Rogers, P. W.; Weber, L.; Beaubien, H. F.; Wilson, J.; Collins, M. Species Identification by Peptide Mass Fingerprinting (PMF) in Fibre Products Preserved by Association with Copper-Alloy Artefacts. J. Archaeol. Sci. 2014, 49, 524– 535,  DOI: 10.1016/j.jas.2014.06.009
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    104
    Species identification by peptide mass fingerprinting (PMF) in fibre products preserved by association with copper-alloy artefacts
    Solazzo, Caroline; Rogers, Penelope Walton; Weber, Leslie; Beaubien, Harriet F.; Wilson, Julie; Collins, Matthew
    Journal of Archaeological Science (2014), 49 (), 524-535CODEN: JASCDU; ISSN:1095-9238. (Elsevier Inc.)
    Fiber products, such as textiles and animal pelts, are often recovered in the corrosion crust of archaeol. metal artifacts. Because clothed burials are an important resource for the study of past societies, accurate fiber identification is important. However, extreme mineralization of animal fibers can render microscopic visualisation difficult for species identification. Peptide mass fingerprinting (PMF) has been successfully used to identify the species origin in both collagen and keratin-made archaeol. artifacts. The approach requires little material but the state of degrdn. (protein hydrolysis) is a limiting factor as it might impact on the identification of key markers. In this study we analyzed pelt and textile fragments found in assocn. with copper-alloy objects with different degrees of mineralization; samples were obtained from a Viking-Age (10th c.) grave in Britain and from a burial in Mongolia (3rd c. BC to 2nd c. AD). Species identification was possible in all but one sample, revealing PMF can be applied to corrosion products, thereby further expanding the value of these objects for textile research.
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  105. 105
    Buckley, M.; Melton, N. D.; Montgomery, J. Proteomics Analysis of Ancient Food Vessel Stitching Reveals > 4000-Year-Old Milk Protein. Rapid Commun. Mass Spectrom. 2013, 27, 531– 538,  DOI: 10.1002/rcm.6481
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    105
    Proteomics analysis of ancient food vessel stitching reveals >4000-year-old milk protein
    Buckley, Michael; Melton, Nigel D.; Montgomery, Janet
    Rapid Communications in Mass Spectrometry (2013), 27 (4), 531-538CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    The 19th century excavation of an exceptionally well-preserved Early Bronze Age high status log-coffin burial from northern England, dated to 2200-2020 BC, yielded a 'food residue' collected from the inside of an accompanying bark vessel. This residue contained fibrous stitching that was used to hold the bark walls of the vessel together, first described as animal sinews, although the surviving material clearly contains animal hairs. Protein sequencing by soft ionization mass spectrometry should identify the proteins that constitute the material, as well as the animal species from which they derive. Peptide mass fingerprinting (PMF) by MALDI-TOF-MS combined with liq. chromatog.-ESI-LTQ-MS/MS was used to identify low-abundance proteins as well as the dominant proteins in the sample. These proteomics techniques revealed the dominant proteins as deriving from the fibrous keratins (both types 1 and 2) and collagens (types 1 and 3), specifically those indicative of a bovine source. However, several peptide sequences diagnostic of bovine α-S1-casein were also obsd., indicating that traces of milk had been preserved within the >4000-yr-old fibrous residue. The presence of this food vessel that once contained milk within a burial of high status is suggestive of the importance placed on these secondary products. It is perhaps more remarkable that this information was retrieved not only from material of such antiquity, but also from an excavation that occurred nearly 200 years ago. Copyright © 2013 John Wiley & Sons, Ltd.
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  106. 106
    Brandt, L. Ø.; Schmidt, A. L.; Mannering, U.; Sarret, M.; Kelstrup, C. D.; Olsen, J. V.; Cappellini, E. Species Identification of Archaeological Skin Objects from Danish Bogs: Comparison between Mass Spectrometry-Based Peptide Sequencing and Microscopy-Based Methods. PLoS One 2014, 9, e106875  DOI: 10.1371/journal.pone.0106875
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    106
    Species identification of archaeological skin objects from Danish bogs: comparison between mass spectrometry-based peptide sequencing and microscopy-based methods
    Brandt, Luise Oersted; Schmidt, Anne Lisbeth; Mannering, Ulla; Sarret, Mathilde; Kelstrup, Christian D.; Olsen, Jesper V.; Cappellini, Enrico
    PLoS One (2014), 9 (9), e106875/1-e106875/10, 10 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Denmark has an extraordinarily large and well-preserved collection of archaeol. skin garments found in peat bogs, dated to approx. 920 BC - AD 775. These objects provide not only the possibility to study prehistoric skin costume and technologies, but also to investigate the animal species used for the prodn. of skin garments. Until recently, species identification of archaeol. skin was primarily performed by light and SEM or the anal. of ancient DNA. However, the efficacy of these methods can be limited due to the harsh, mostly acidic environment of peat bogs leading to morphol. and mol. degrdn. within the samples. We compared species assignment results of twelve archaeol. skin samples from Danish bogs using Mass Spectrometry (MS)-based peptide sequencing, against results obtained using light and SEM. While it was difficult to obtain reliable results using microscopy, MS enabled the identification of several species-diagnostic peptides, mostly from collagen and keratins, allowing confident species discrimination even among taxonomically close organisms, such as sheep and goat. Unlike previous MS-based methods, mostly relying on peptide fingerprinting, the shotgun sequencing approach we describe aims to identify the complete extd. ancient proteome, without preselected specific targets. As an example, we report the identification, in one of the samples, of two peptides uniquely assigned to bovine fetal Hb, indicating the prodn. of skin from a calf slaughtered within the first months of its life. We conclude that MS-based peptide sequencing is a reliable method for species identification of samples from bogs. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium with the dataset identifier PXD001029.
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  107. 107
    Yang, Y.; Shevchenko, A.; Knaust, A.; Abuduresule, I.; Li, W.; Hu, X.; Wang, C.; Shevchenko, A. Proteomics Evidence for Kefir Dairy in Early Bronze Age China. J. Archaeol. Sci. 2014, 45, 178– 186,  DOI: 10.1016/j.jas.2014.02.005
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    107
    Proteomics evidence for kefir dairy in Early Bronze Age China
    Yang, Yimin; Shevchenko, Anna; Knaust, Andrea; Abuduresule, Idelisi; Li, Wenying; Hu, Xingjun; Wang, Changsui; Shevchenko, Andrej
    Journal of Archaeological Science (2014), 45 (), 178-186CODEN: JASCDU; ISSN:1095-9238. (Elsevier Inc.)
    Cheese making has been inferred at several sites in northern Europe as early as the 6th millennium BC and was common in Egypt and Mesopotamia in 3rd millennium BC. However, the remains of ancient cheeses have never been found and recipes of ancient dairy, its prodn. scale, social and economic impact remain poorly understood. Here we present direct proteomics evidence for the prodn. of an earliest known cheese that was found as an org. mass assocd. with the mummies of Early Bronze Age cemetery of Xiaohe (1980-1450 BC) in Xinjiang, China. Kefir fermn. of ruminant milk by a symbiotic culture of Lactobacillus kefiranofaciens and other lactic acid bacteria and yeasts was the basis of robust, scalable, probiotic, lactose-free dairy and a key technol. advance that introduced economic benefits of extensive herding into a semi-pastoral household of the Eastern Eurasia population already in the Early Bronze Age.
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  108. 108
    Xie, M.; Shevchenko, A.; Wang, B.; Shevchenko, A.; Wang, C.; Yang, Y. Identification of a Dairy Product in the Grass Woven Basket from Gumugou Cemetery (3800 BP, Northwestern China). Quat. Int. 2016, 426, 158– 165,  DOI: 10.1016/j.quaint.2016.04.015
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    Hollemeyer, K.; Altmeyer, W.; Heinzle, E.; Pitra, C. Species Identification of Oetzi’s Clothing with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Based on Peptide Pattern Similarities of Hair Digests. Rapid Commun. Mass Spectrom. 2008, 22, 2751– 2767,  DOI: 10.1002/rcm.3679
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    109
    Species identification of Oetzi's clothing with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on peptide pattern similarities of hair digests
    Hollemeyer, Klaus; Altmeyer, Wolfgang; Heinzle, Elmar; Pitra, Christian
    Rapid Communications in Mass Spectrometry (2008), 22 (18), 2751-2767CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Identification of ancient biol. samples from the 1991-discovered and more than 5300-yr-old Tyrolean mummy, also called iceman or Oetzi, is very difficult. The species of origins of four animal-hair-bearing samples of the accoutrement of the mummy not yet diagnosed were identified by a special proteomics method. Ha 43/91/130 and Ha 6/91, two samples from his coat, and Ha 5/91, a sample from his leggings, were assigned to sheep. The upper leather of his moccasins, Ha 2/91, was made from cattle. Despite the enormous age of these samples with partial (bio)chem. alterations, reliable identification was possible using a recently developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometric ((MALDI-TOF MS)-based anal. method. The method is exclusively based on the anal. of proteins and uses minute amts. of peptides directly derived from tryptic hair digests without any sepn. or enrichment steps. Unknown species are identified by comparison of their peptide ion patterns with known spectra stored in existing databases. Hereby, the correlation distance, a form of Euclidean distance, and deduced parameters are used to measure similarities. If more than one potential hit remains, specific diagnostic peptide ions are used to stepwise exclude incorrect matches. These ions are specific for orders, families, subfamilies/genera and/or even species. Peptide mass fingerprinting data combined with those from collision-induced dissocn. spectra (combined MS & MS/MS) were used for interpretation with the MASCOT search engine and the NCBI database to find the potential parentage of hair proteins. For this technique, selected precursor ions were identified as specific diagnostic peptide ions.
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    Barker, A.; Dombrosky, J.; Venables, B.; Wolverton, S. Taphonomy and Negative Results: An Integrated Approach to Ceramic-Bound Protein Residue Analysis. J. Archaeol. Sci. 2018, 94, 32– 43,  DOI: 10.1016/j.jas.2018.03.004
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    Taphonomy and negative results: An integrated approach to ceramic-bound protein residue analysis
    Barker, Andrew; Dombrosky, Jonathan; Venables, Barney; Wolverton, Steve
    Journal of Archaeological Science (2018), 94 (), 32-43CODEN: JASCDU; ISSN:1095-9238. (Elsevier Inc.)
    Despite the growing body of evidence demonstrating that proteins can survive for thousands to even millions of years in selected contexts, there are relatively few examples of the successful recovery and identification of archaeol. protein residues from ceramic artifacts. Claims of pos. results are sometimes contentious and frequently challenged. One source of confusion in the debate is a general lack of consideration for the taphonomic histories of ceramic-bound proteins. To gain insight into this issue, we conducted an integrated, mass spectrometry-based study examg. ceramic-bound protein that was exptl. aged over the course of 12 mo. Results demonstrate the rapid degrdn. of proteins, raise questions about the degree to which ceramic-bound proteins can be expected to survive over time, and reveal some of the limitations of non-targeted mass spectrometry-based analyses. Further, by comparing results from our exptl.-aged samples to the those we obtained from a multi-pronged study of archaeol. ceramics from the American Southwest, we are able to draw more confident conclusions regarding our lack of meaningful matches in the archaeol. samples.
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    Eisele, J. A.; Fowler, D. D.; Haynes, G.; Lewis, R. A. Survival and Detection of Blood Residues on Stone Tools. Antiquity 1995, 69, 36– 46,  DOI: 10.1017/S0003598X00064280
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    Leach, J. D.; Mauldin, R. P. Additional Comments on Blood Residue Analysis in Archaeology. Antiquity 1995, 69, 1020– 1022,  DOI: 10.1017/S0003598X00082570
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    Cattaneo, C.; Gelsthorpe, K.; Phillips, P.; Sokol, R. J. Blood Residues on Stone Tools: Indoor and Outdoor Experiments. World Archaeol. 1993, 25, 29– 43,  DOI: 10.1080/00438243.1993.9980226
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    Blood residues on stone tools: indoor and outdoor experiments
    Cattaneo C; Gelsthorpe K; Phillips P; Sokol R J
    World archaeology (1993), 25 (1), 29-43 ISSN:0043-8243.
    There is no expanded citation for this reference.
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    Covington, A. D. Modern Tanning Chemistry. Chem. Soc. Rev. 1997, 26, 111– 126,  DOI: 10.1039/cs9972600111
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    Modern tanning chemistry
    Covington, Anthony D.
    Chemical Society Reviews (1997), 26 (2), 111-126CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review with 30 refs. on chem. technologies used for tanning leather; traditional methods of tanning are explained and newer processes are described. The areas of tanning include: vegetable tanning with plant polyphenols, mineral tanning with metal salts, in particular chromium(III), oil and aldehyde tannages, synthetic tanning agents and org. tannages based on natural polyphenols or synthetic org. oligomers. The fundamental nature of the tanning reaction and the origin of hydrothermal stability are discussed.
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    Yu, T.-Y.; Morton, J. D.; Clerens, S.; Dyer, J. M. Cooking-Induced Protein Modifications in Meat. Compr. Rev. Food Sci. 2017, 16, 141– 159,  DOI: 10.1111/1541-4337.12243
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    Cooking-Induced Protein Modifications in Meat
    Yu, Tzer-Yang; Morton, James D.; Clerens, Stefan; Dyer, Jolon M.
    Comprehensive Reviews in Food Science and Food Safety (2017), 16 (1), 141-159CODEN: CRFSBJ; ISSN:1541-4337. (Institute of Food Technologists)
    A review. Food ingredients commonly undergo heat treatment. Meat, in particular, is typically consumed after some form of heating, such as boiling or roasting. Heating of meat can introduce a wide range of structural changes in its proteinaceous components. At the 3-dimensional structural level, meat proteins may denature and form aggregates upon heating. At the mol. level, primary structure (amino acid residue) alterations reported in cooked meat include protein carbonylation, modification of arom. residues, and the formation of Maillard reaction products. Identification of these modifications is essential for detg. the mechanism of thermal processing of meat and allowing better control of the nutritional and functional properties of products. This article reviews and summarizes the current state of understanding of protein modifications at the mol. level in commonly consumed mammalian food. In addn., relevant case studies relating to characterization of heat-induced amino acid residue-level modifications in other biol. materials such as milk and wool are discussed to provide complementary insights.
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    Gil-Bona, A.; Bidlack, F. B. Tooth Enamel and Its Dynamic Protein Matrix. Int. J. Mol. Sci. 2020, 21, 4458,  DOI: 10.3390/ijms21124458
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    Tooth enamel and its dynamic protein matrix
    Gil-Bona, Ana; Bidlack, Felicitas B.
    International Journal of Molecular Sciences (2020), 21 (12), 4458CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. Tooth enamel is the outer covering of tooth crowns, the hardest material in the mammalian body, yet fracture resistant. The extremely high content of 95%wt. calcium phosphate in healthy adult teeth is achieved through mineralization of a proteinaceous matrix that changes in abundance and compn. Enamel-specific proteins and proteases are known to be crit. for proper enamel formation. Recent proteomics analyses revealed many other proteins with their roles in enamel formation yet to be unraveled. Although the exact protein compn. of healthy tooth enamel is still unknown, it is apparent that compromised enamel deviates in amt. and compn. of its org. material. Why these differences affect both the mineralization process before tooth eruption and the properties of erupted teeth will become apparent as proteomics protocols are adjusted to the variability between species, tooth size, sample size and ephemeral org. content of forming teeth. This review summarizes the current knowledge and published proteomics data of healthy and diseased tooth enamel, including advancements in forensic applications and disease models in animals. A summary and discussion of the status quo highlights how recent proteomics findings advance our understating of the complexity and temporal changes of extracellular matrix compn. during tooth enamel formation.
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    Hedges, J. I. Global Giogeochemical Cycles: Progress and Problems. Mar. Chem. 1992, 39, 67– 93,  DOI: 10.1016/0304-4203(92)90096-S
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    Global biogeochemical cycles: progress and problems
    Hedges, John I.
    Marine Chemistry (1992), 39 (1-3), 67-93CODEN: MRCHBD; ISSN:0304-4203.
    A review with many refs. on the large-scale processes through which bioactive substances are stored and exchanged among modern and geol. reservoirs. Org. compds. and the role of the ocean are particularly stressed.
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    Benbow, M. E.; Barton, P. S.; Ulyshen, M. D.; Beasley, J. C.; DeVault, T. L.; Strickland, M. S.; Tomberlin, J. K.; Jordan, H. R.; Pechal, J. L. Necrobiome Framework for Bridging Decomposition Ecology of Autotrophically and Heterotrophically Derived Organic Matter. Ecol. Monogr. 2019, 89, e01331  DOI: 10.1002/ecm.1331
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    Solazzo, C.; Dyer, J. M.; Clerens, S.; Plowman, J.; Peacock, E. E.; Collins, M. J. Proteomic Evaluation of the Biodegradation of Wool Fabrics in Experimental Burials. Int. Biodeterior. Biodegradation 2013, 80, 48– 59,  DOI: 10.1016/j.ibiod.2012.11.013
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    Proteomic evaluation of the biodegradation of wool fabrics in experimental burials
    Solazzo, Caroline; Dyer, Jolon M.; Clerens, Stefan; Plowman, Jeff; Peacock, Elizabeth E.; Collins, Matthew J.
    International Biodeterioration & Biodegradation (2013), 80 (), 48-59CODEN: IBBIES; ISSN:0964-8305. (Elsevier Ltd.)
    Woollen textiles recovered from archaeol. excavations are usually fragile, discolored, mineralized, or highly biodeteriorated fragmentary remains. The nature and extent of preservation is highly dependent on the site of burial and factors such as soil compn., pH, temp., oxygen content, and contact with a wood coffin or metals. Understanding the particular biodegrdn. in archaeol. sites is important for biomol. studies of textiles, and to assist in the conservation of these finds. Wool fabrics dyed and buried for up to 8 yr in bog-type soils in Denmark (Lejre) and Norway (Rormyra), and in marine sediments in Sweden (Marstrand) were evaluated by proteomics anal. Wool degrdn. was found to occur through a range of differing mechanisms, mainly due to the complex nature of wool itself with its many families of proteins (keratin and keratin-assocd. proteins) and structures. Microbial activity was a large contributory factor to the phys. deterioration of the wool fabrics at Lejre and Marstrand, and might result in faster loss of keratin-assocd. proteins over keratins. Addnl. hydrolysis took place at Marstrand, influenced by the environmental conditions of the sediment, and in particular the alk. pH, contributing to the degrdn. of keratins. However, crosslinking was assocd. with the long-term preservation of the fabrics at Rormyra, where pH, temp., and vegetative compn. of the bog prevented microbial activity, and sphagnum moss might preserve wool by binding with keratins.
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  120. 120
    Saitta, E. T.; Rogers, C.; Brooker, R. A.; Abbott, G. D.; Kumar, S.; O’Reilly, S. S.; Donohoe, P.; Dutta, S.; Summons, R. E.; Vinther, J. Low Fossilization Potential of Keratin Protein Revealed by Experimental Taphonomy. Palaeontology 2017, 60, 547– 556,  DOI: 10.1111/pala.12299
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    Wadsworth, C.; Buckley, M. Proteome Degradation in Fossils: Investigating the Longevity of Protein Survival in Ancient Bone. Rapid Commun. Mass Spectrom. 2014, 28, 605– 615,  DOI: 10.1002/rcm.6821
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    121
    Proteome degradation in fossils: investigating the longevity of protein survival in ancient bone
    Wadsworth, Caroline; Buckley, Mike
    Rapid Communications in Mass Spectrometry (2014), 28 (6), 605-615CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    We report the use of proteomics techniques to study how the fossil bone proteome changes in complexity over one million years. METHODS: We include the attempted use of a previously unreported methodol. in proteome research, to remove the dominant bone collagens using bacterial collagenase as well as conventional shotgun proteomics methodol. following digestion with the protease trypsin. In this study we expand upon a set of 19 bovine sub-fossil specimens ranging over one and a half million years that had previously been shown to possess collagen, using a total of 46 LTQ-Orbitrap liq. chromatog./tandem mass spectrometry (LC/MS/MS) analyses contg. 462,186 precursor ion analyses. RESULTS: Although many types of proteins can typically be identified in recent bone, in degraded bone we observe a rapid loss of lower abundance proteins. Abundant serum proteins such as serum albumin and alpha-2-HS-glycoprotein appear to be more easily recovered in ancient bone, both being identified in specimens dating to the Early Pleistocene, the earliest period tested in this study. Proteins belonging to the leucine-rich repeat family such as lumican, biglycan and chondroadherin also survive well, possibly because of their interactions with bone collagen. CONCLUSIONS: Of these 'survivor proteins' A2HSG shows a remarkable amt. of sequence variation, making it potentially one of the most useful proteins to study for species identification and phylogenetic inference in archaeol. and palaeontol. bone. © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.
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  122. 122
    Brownlow, S.; Morais Cabral, J. H.; Cooper, R.; Flower, D. R.; Yewdall, S. J.; Polikarpov, I.; North, A. C. T.; Sawyer, L. Bovine β-Lactoglobulin at 1.8 Å Resolution ─ Still an Enigmatic Lipocalin. Structure 1997, 5, 481– 495,  DOI: 10.1016/S0969-2126(97)00205-0
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    Bovine β-lactoglobulin at 1.8 Å resolution - still an enigmatic lipocalin
    Brownlow, Sharon; Cabral, Joao H. Morais; Cooper, Ron; Flower, Darren R.; Yewdall, Stephen J.; Polikarpov, Igor; North, Anthony Ct; Sawyer, Lindsay
    Structure (London) (1997), 5 (4), 481-495CODEN: STRUE6; ISSN:0969-2126. (Current Biology)
    β-Lactoglobulin (I) is the major whey protein in the milk of ruminants and many other mammals. Its function is not known, but it undergoes at least 2 pH-dependent conformational changes which may be important. Bovine I crystallizes in several different lattices, and medium-resoln. structures of orthorhombic lattice Y and trigonal lattice Z have previously been published. Triclinic lattice X and lattice Z crystals grow at pH values either side of the pH at which one of the pH-induced conformational changes occurs. A full understanding of the structure is needed to help explain both the conformational changes and the different denaturation behavior of the genetic variants. Here, the authors redetd. the structure of I lattice Z at 3.0 Å resoln. by multiple isomorphous replacement and partially refined it (R factor = 24.8%). Using the dimer from this lattice Z structure as a search model, the triclinic crystal form grown at pH 6.5 (lattice X) was solved by mol. replacement. Refinement of lattice X at 1.8 Å resoln. gave an R factor of 18.1%. The crystal structure that was detd. differed from previously published structures in several ways. Incorrect threading of the sequence in the published structures of I affected 4 of the 9 β-strands. The basic lipocalin fold of the polypeptide chain was unchanged, however. The relative orientation of the monomers in the I dimer differed in the 2 lattices. On raising the pH, there was a rotation of ∼5°, which broke a no. of intersubunit H-bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the β-strand with the free thiol, each of which shows genetic variation.
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    Pekar, J.; Ret, D.; Untersmayr, E. Stability of Allergens. Mol. Immunol. 2018, 100, 14– 20,  DOI: 10.1016/j.molimm.2018.03.017
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    Stability of allergens
    Pekar, Judith; Ret, Davide; Untersmayr, Eva
    Molecular Immunology (2018), 100 (), 14-20CODEN: MOIMD5; ISSN:0161-5890. (Elsevier)
    A review. For proteins to cause IgE-mediated allergic reactions, several common characteristics have to be defined, including small mol. size, soly. and stability to changing pH levels and enzymic degrdn. Nevertheless, these features are not unique for potent allergens, but are also obsd. in non-allergenic proteins. Due to the increasing awareness by regulatory authorities regarding the allergy pandemic, definition of characteristics unique to potent allergens would facilitate allergenicity assessment in the future. Despite major research efforts even to date the features unique for major allergens have not been elucidated so far. The route of allergen entry into the organism dets. to a great extent these required characteristics. Esp. orally ingested allergens are exposed to the harsh milieu of the gastrointestinal tract but might addnl. be influenced by food processing. Depending on mol. properties such as disulfide bonds contributing to protein fold and formation of conformational IgE epitopes, posttranslational protein modification or protein food matrix interactions, enzymic and thermal stability might differ between allergens. Moreover, also ligand binding influences structural stability. In the current review article, we aim at highlighting specific characteristics and mol. pattern contributing to a stabilized protein structure and overall allergenicity.
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    Procopio, N.; Williams, A.; Chamberlain, A. T.; Buckley, M. Forensic Proteomics for the Evaluation of the Post-Mortem Decay in Bones. J. Proteomics 2018, 177, 21– 30,  DOI: 10.1016/j.jprot.2018.01.016
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    Forensic proteomics for the evaluation of the post-mortem decay in bones
    Procopio, Noemi; Williams, Anna; Chamberlain, Andrew T.; Buckley, Michael
    Journal of Proteomics (2018), 177 (), 21-30CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)
    Current methods for evaluation the of post-mortem interval (PMI) of skeletal remains suffer from poor accuracy due to the great no. of variables that affect the diagenetic process and to the lack of specific guidelines to address this issue. During decompn., proteins can undergo cumulative decay over the time, resulting in a decrease in the range and abundance of proteins present (i.e., the proteome) in different tissues as well as in an increase of post-translational modifications occurring in these proteins. In this study, we investigate the applicability of bone proteomic analyses to simulated forensic contexts, looking for specific biomarkers that may help the estn. of PMI, as well as evaluate a previously discovered marker for the estn. of biol. age. We noticed a redn. of particular plasma and muscle proteins with increasing PMIs, as well as an increased deamidation of biglycan, a protein with a role in modulating bone growth and mineralization. We also corroborated our previous results regarding the use of fetuin-A as a potential biomarker for the estn. of age-at-death, demonstrating the applicability and the great potential that proteomics may have towards forensic sciences. The estn. of the post-mortem interval has a key role in forensic investigations, however nowadays it still suffers from poor reliability, esp. when body tissues are heavily decompd. Here we propose for the first time the application of bone proteomics to the estn. of the time elapsed since death and found several new potential biomarkers to address this, demonstrating the applicability of proteomic analyses to forensic sciences.
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    Kendall, C.; Eriksen, A. M. H.; Kontopoulos, I.; Collins, M. J.; Turner-Walker, G. Diagenesis of Archaeological Bone and Tooth. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2018, 491, 21– 37,  DOI: 10.1016/j.palaeo.2017.11.041
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    Demarchi, B. Amino Acids and Proteins in Fossil Biominerals: An Introduction for Archaeologists and Palaeontologists; John Wiley & Sons, 2020.  DOI: 10.1002/9781119089537 .
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    Cleland, T. P.; Schroeter, E. R.; Colleary, C. Diagenetiforms: A New Term to Explain Protein Changes as a Result of Diagenesis in Paleoproteomics. J. Proteomics 2021, 230, 103992,  DOI: 10.1016/j.jprot.2020.103992
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    Diagenetiforms: A new term to explain protein changes as a result of diagenesis in paleoproteomics
    Cleland, Timothy P.; Schroeter, Elena R.; Colleary, Caitlin
    Journal of Proteomics (2021), 230 (), 103992CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)
    A review. The term proteoform describes all combinations of change in a protein, as elucidated through intact mass proteomics. Paleoproteomic studies have begun using digestion-free and top-down techniques to access information from ancient and historical remains. However, to discuss protein changes that uniquely occur to archaeol. and paleontol. proteomes as the result of diagenesis (i.e., phys. and chem. change imparted by burial), a novel term is needed that both addresses issues of combinatorics and distinguishes diagenetic-specific alteration. The term diagenetiform provides the opportunity to communicate clearly the sets of diagenetic changes found on preserved proteins. The diagenetiform nomenclature will allow for top-down paleoproteomic studies to accurately describe the total changes detected on ancient proteins.
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    Cleland, T. P.; Schroeter, E. R.; Schweitzer, M. H. Biologically and Diagenetically Derived Peptide Modifications in Moa Collagens. Proc. Biol. Sci. 2015, 282, 20150015,  DOI: 10.1098/rspb.2015.0015
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    Biologically and diagenetically derived peptide modifications in moa collagens
    Cleland Timothy P; Schroeter Elena R; Schweitzer Mary H
    Proceedings. Biological sciences (2015), 282 (1808), 20150015 ISSN:.
    The modifications that occur on proteins in natural environments over time are not well studied, yet characterizing them is vital to correctly interpret sequence data recovered from fossils. The recently extinct moa (Dinornithidae) is an excellent candidate for investigating the preservation of proteins, their post-translational modifications (PTMs) and diagenetic alterations during degradation. Moa protein extracts were analysed using mass spectrometry, and peptides from collagen I, collagen II and collagen V were identified. We also identified biologically derived PTMs (i.e. methylation, di-methylation, alkylation, hydroxylation, fucosylation) on amino acids at locations consistent with extant proteins. In addition to these in vivo modifications, we detected novel modifications that are probably diagenetically derived. These include loss of hydroxylation/glutamic semialdehyde, carboxymethyllysine and peptide backbone cleavage, as well as previously noted deamidation. Moa collagen sequences and modifications provide a baseline by which to evaluate proteomic studies of other fossils, and a framework for defining the molecular relationship of moa to other closely related taxa.
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    Oldenburg, T.; Brown, M.; Inwood, J.; Radović, J.; Snowdon, R.; Larter, S.; Mercader, J. A Novel Route for Identifying Starch Diagenetic Products in the Archaeological Record. PLoS One 2021, 16, e0258779  DOI: 10.1371/journal.pone.0258779
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    129
    A novel route for identifying starch diagenetic products in the archaeological record
    Oldenburg, Thomas; Brown, Melisa; Inwood, Jamie; Radovic, Jagos; Snowdon, Ryan; Larter, Steve; Mercader, Julio
    PLoS One (2021), 16 (11), e0258779CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    This work introduces a novel anal. chem. method potentially applicable to the study of archaeol. starch residues. The investigation involved the lab. synthesis of model Maillard reaction mixts. and their anal. through Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS). Thus, starch from sixteen plant species were matured while reacting it with the amino acid glycine. The FTICR-MS anal. revealed > 5,300 mol. compds., with numerous unique heteroatom rich compd. classes, ranging from 20 (Zea mays) to 50 (Sorghum bicolor). These classes were investigated as repositories of chem. structure retaining source and process-specific character, linked back to botanical provenance. We discussed the Maillard reaction products thus generated, a possible pathway for the preservation of degraded starch, while also assessing diagenetic recalcitrance and adsorption potential to mineral surfaces. In some cases, hydrothermal experimentation on starches without glycine reveals that the chem. complexity of the starch itself is sufficient to produce some Maillard reaction products. The article concludes that FTICR-MS offers a new anal. window to characterize starchy residue and its diagenetic products, and is able to recognize taxonomic signals with the potential to persist in fossil contexts.
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  130. 130
    Collins, M. J.; Bishop, A. N.; Farrimond, P. Sorption by Mineral Surfaces: Rebirth of the Classical Condensation Pathway for Kerogen Formation?. Geochim. Cosmochim. Acta 1995, 59, 2387– 2391,  DOI: 10.1016/0016-7037(95)00114-F
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    130
    Sorption by mineral surfaces: rebirth of the classical condensation pathway for kerogen formation?
    Collins, Matthew J.; Bishop, Andrew N.; Farrimond, Paul
    Geochimica et Cosmochimica Acta (1995), 59 (11), 2387-91CODEN: GCACAK; ISSN:0016-7037. (Elsevier)
    R.G. Keil et al. (1994) and L.M. Mayer (1994a,b) both found a strong correlation between surface area and org. content in marine sediments. This finding suggests that occurrence of org. matter in marine sediments typically is as a monolayer (or equiv. concn.) sorbed to the surface of mineral grains. Theor. factors which may influence the adsorption and propagation of polymeric org. matter on mineral surfaces are considered and the likely diagenetic fate of adsorbed material is examd. Both adsorption and condensation have been suggested as possible mechanisms for the preservation of labile biopolymers, but neither process is satisfactory as a stand-alone mechanism; adsorption of monomers can merely retard their biodegrdn., and condensation is not favored in soln. However, if the two processes operate in concert, the criticisms leveled against each process considered in isolation are cancelled out, adsorption promoting condensation and condensation enhancing the strength of adsorption of the products. The authors suggest that the diagenetic modifications of surface adsorbed org. mols. will tend to strengthen their binding to the mineral surface, such that the geomacromols. will evolve on the mineral surface towards strongly bound monolayers. The hypothesis overcomes many of the objections to the so-called classical condensation pathway of kerogen formation.
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  131. 131
    Walton, D. Degradation of Intracrystalline Proteins and Amino Acids in Fossil Brachiopods. Org. Geochem. 1998, 28, 389– 410,  DOI: 10.1016/S0146-6380(97)90126-1
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    131
    Degradation of intracrystalline proteins and amino acids in fossil brachiopods
    Walton, Derek
    Organic Geochemistry (1998), 28 (6), 389-410CODEN: ORGEDE; ISSN:0146-6380. (Elsevier Science Ltd.)
    Four genera of Recent to Plio-Pleistocene articulated brachiopods were collected from up to 16 horizons spanning the last 3.3 Ma of sediment deposition in the South Wanganui Basin, New Zealand, and assayed for the preservation of intracryst. proteins and/or amino acids. The proteins present in the shells of living and Recent brachiopods undergo rapid degrdn. through the decompn. of the peptide bond. Up to 95% of the constituent amino acids from the proteins are present in the free state by 0.12 Ma. This rate of degrdn. is far higher than was originally expected for intracryst. proteins. Quant. anal. of the concns. of amino acids present within the shells of fossil brachiopods indicates a range of reaction rates for the subsequent degrdn. of individual amino acids. The degrdn. of these amino acids may lead to the total loss of compds., to the generation of non-std. amino acids, or to diagenetically produced proteinaceous amino acids. These reactions do not necessarily mirror those which occur during the pyrolysis of an aq. soln. of the pure amino acids, either in their rate or products.
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    Buckley, M.; Wadsworth, C. Proteome Degradation in Ancient Bone: Diagenesis and Phylogenetic Potential. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2014, 416, 69– 79,  DOI: 10.1016/j.palaeo.2014.06.026
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    Craig, O. E.; Collins, M. J. The Removal of Protein from Mineral Surfaces: Implications for Residue Analysis of Archaeological Materials. J. Archaeol. Sci. 2002, 29, 1077– 1082,  DOI: 10.1006/jasc.2001.0757
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    Carrera, M. Proteomics and Food Analysis: Principles, Techniques, and Applications. Foods 2021, 10, 2538,  DOI: 10.3390/foods10112538
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    Keller, B. O.; Sui, J.; Young, A. B.; Whittal, R. M. Interferences and Contaminants Encountered in Modern Mass Spectrometry. Anal. Chim. Acta 2008, 627, 71– 81,  DOI: 10.1016/j.aca.2008.04.043
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    Interferences and contaminants encountered in modern mass spectrometry
    Keller, Bernd O.; Sui, Jie; Young, Alex B.; Whittal, Randy M.
    Analytica Chimica Acta (2008), 627 (1), 71-81CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    A review. With the invention of electrospray ionization and matrix-assisted laser desorption/ionization, scientists employing modern mass spectrometry naturally face new challenges with respect to background interferences and contaminants that might not play a significant role in traditional or other anal. techniques. Efforts to continuously minimize sample vols. and measurable concns. increase the need to understand where these interferences come from, how they can be identified, and if they can be eliminated. Knowledge of identity enables their use as internal calibrants for accurate mass measurements. This review/tutorial summarizes current literature on reported contaminants and introduces a no. of novel interferences that have been obsd. and identified in our labs. over the past decade. These include both compds. of proteinaceous and non-proteinaceous nature. In the supplemental data a spreadsheet is provided that contains a searchable ion list of all compds. identified to date.
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  136. 136
    Schroeter, E. R.; DeHart, C. J.; Schweitzer, M. H.; Thomas, P. M.; Kelleher, N. L. Bone Protein “Extractomics”: Comparing the Efficiency of Bone Protein Extractions of Gallus Gallus in Tandem Mass Spectrometry, with an Eye Towards Paleoproteomics. PeerJ. 2016, 4, e2603  DOI: 10.7717/peerj.2603
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    Mackie, M.; Rüther, P.; Samodova, D.; Di Gianvincenzo, F.; Granzotto, C.; Lyon, D.; Peggie, D. A.; Howard, H.; Harrison, L.; Jensen, L. J. Palaeoproteomic Profiling of Conservation Layers on a 14th Century Italian Wall Painting. Angew. Chem., Int. Ed. Engl. 2018, 57, 7369– 7374,  DOI: 10.1002/anie.201713020
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    137
    Palaeoproteomic Profiling of Conservation Layers on a 14th Century Italian Wall Painting
    Mackie Meaghan; Di Gianvincenzo Fabiana; Granzotto Clara; Cappellini Enrico; Mackie Meaghan; Ruther Patrick; Samodova Diana; Olsen Jesper V; Lyon David; Jensen Lars Juhl; Peggie David A; Howard Helen; Harrison Lynne
    Angewandte Chemie (International ed. in English) (2018), 57 (25), 7369-7374 ISSN:.
    Ahead of display, a non-original layer was observed on the surface of a fragment of a wall painting by Ambrogio Lorenzetti (active 1319, died 1348/9). FTIR analysis suggested proteinaceous content. Mass spectrometry was used to better characterise this layer and revealed two protein components: sheep and cow glue and chicken and duck egg white. Analysis of post-translational modifications detected several photo-oxidation products, which suggest that the egg experienced prolonged exposure to UV light and was likely applied long before the glue layer. Additionally, glycation products detected may indicate naturally occurring glycoprotein degradation or reaction with a carbohydrate material such as starch, identified by ATR-FTIR in a cross-section of a sample taken from the painting. Palaeoproteomics is shown to provide detailed characterisation of organic layers associated with mural paintings and therefore aids reconstruction of the conservation history of these objects.
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  138. 138
    Cleland, T. P.; Voegele, K.; Schweitzer, M. H. Empirical Evaluation of Bone Extraction Protocols. PLoS One 2012, 7, e31443  DOI: 10.1371/journal.pone.0031443
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    138
    Empirical evaluation of bone extraction protocols
    Cleland, Timothy P.; Voegele, Kristyn; Schweitzer, Mary H.
    PLoS One (2012), 7 (2), e31443CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    The application of high-resoln. anal. techniques to characterize ancient bone proteins requires clean, efficient extn. to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and relative purity using the identification of antibody-antigen complexes on ELISA and gel electrophoresis. Moa bone provided an ancient comparison for the effectiveness of bone extn. protocols tested on ostrich bone. For the immunol. part of this study, we focused on collagen I, osteocalcin, and Hb because collagen and osteocalcin are the most abundant proteins in the mineralized extracellular matrix and Hb is common in the vasculature. Most of these procedures demineralize the bone first, and then the remaining orgs. are chem. extd. We found that the use of hydrochloric acid, rather than EDTA, for demineralization resulted in the cleanest extns. because the acid was easily removed. In contrast, the use of EDTA resulted in smearing upon electrophoretic sepn., possibly indicating these samples were not as pure. The denaturing agents sodium dodecyl sulfate, urea, and guanidine HCl have been used extensively for the solubilization of proteins in non-biomineralized tissue, but only the latter has been used on bone. We show that all three denaturing agents are effective for extg. bone proteins. One addnl. method tested uses ammonium bicarbonate as a solubilizing buffer that is more appropriate for post-extn. analyses (e.g., proteomics) by removing the need for desalting. We found that both guanidine HCl and ammonium bicarbonate were effective for extg. many bone proteins, resulting in similar electrophoretic patterns. With the increasing use of proteomics, a new generation of scientists are now interested in the study of proteins from not only extant bone but also from ancient bone.
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  139. 139
    Wang, N.; Brown, S.; Ditchfield, P.; Hebestreit, S.; Kozilikin, M.; Luu, S.; Wedage, O.; Grimaldi, S.; Chazan, M.; Kolska, L. H. Testing the Efficacy and Comparability of ZooMS Protocols on Archaeological Bone. J. Proteomics 2021, 233, 104078,  DOI: 10.1016/j.jprot.2020.104078
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    Procopio, N.; Buckley, M. Minimizing Laboratory-Induced Decay in Bone Proteomics. J. Proteome Res. 2017, 16, 447– 458,  DOI: 10.1021/acs.jproteome.6b00564
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    140
    Minimizing Laboratory-Induced Decay in Bone Proteomics
    Procopio, Noemi; Buckley, Michael
    Journal of Proteome Research (2017), 16 (2), 447-458CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Proteomics methods are being increasingly used to study archeol. and palaeontol. bone, assisting in species identification and phylogenetic studies as well as improving the understanding of bone diagenesis. More recently, there are developing interests in the study of post-translation modifications (PTMs), some of which are potentially diagnostic of decay, but none of the previous extn. methods have been developed in light of this. To be able to record close to natural deamidation levels of samples an extn. procedure should minimize lab.-induced decay, such as asparagine and glutamine deamidations, which are considered most strongly related with decay and known to occur frequently with std. lab. procedures. Here the authors tested numerous methods to identify an optimal approach of extg. proteins from bone while minimizing artificial decay. Using a weak acid to partially demineralize the bone sample, then subsequent incubation of the acid insol. fraction with guanidine hydrochloride and enzymic digestion in ammonium acetate, the authors obsd. a ∼50% redn. in deamidation while also substantially decreasing the protocol length. The authors propose this optimized method as appropriate for studies of archeol., palaeontol. as well as potentially forensic studies using proteomics where decay measurements could act as "mol. timers".
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    Procopio, N.; Chamberlain, A. T.; Buckley, M. Exploring Biological and Geological Age-Related Changes through Variations in Intra- and Intertooth Proteomes of Ancient Dentine. J. Proteome Res. 2018, 17, 1000– 1013,  DOI: 10.1021/acs.jproteome.7b00648
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    141
    Exploring Biological and Geological Age-related Changes through Variations in Intra- and Intertooth Proteomes of Ancient Dentine
    Procopio, Noemi; Chamberlain, Andrew T.; Buckley, Michael
    Journal of Proteome Research (2018), 17 (3), 1000-1013CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Proteomic analyses are becoming more widely used in archeol. not only due to the greater preservation of proteins in ancient specimens than DNA but also because they can offer different information, particularly relating to compositional preservation and potentially a means to est. biol. and geol. age. However, it remains unclear to what extent different burial environments impact these aspects of proteome decay. Teeth have to date been much less studied than bone but are ideal to explore how proteins decay with time due to the negligible turnover that occurs in dentin relative to bone. We investigated the proteome variability and deamidation levels of different sections of molar teeth from archeol. bovine mandibles as well as their mandibular bone. We obtained a greater yield of proteins from the crown of the teeth but did not find differences between the different molars analyzed within each mandible. We also obtained the best variety of protein from a well-preserved mandible that was not the youngest one in terms of chronol. age, showing the influence of the preservation conditions on the final proteomic outcome. Intriguingly, we also noticed an increase in abundance levels of fetuin-A in biol. younger mandibles as reported previously, but the opposite trend in tooth dentin. Interestingly, we obsd. higher glutamine deamidation levels in teeth from the geol. oldest mandible despite it being the biol. youngest specimen, showing that the archeol. age strongly impacts on the level of deamidations obsd., much more so than biol. aging. This indicates that the glutamine deamidation ratio of selected peptides may act as a good predictor of the relative geochronol. age of archeol. specimens.
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  142. 142
    Procopio, N.; Hopkins, R. J. A.; Harvey, V. L.; Buckley, M. Proteome Variation with Collagen Yield in Ancient Bone. J. Proteome Res. 2021, 20, 1754– 1769,  DOI: 10.1021/acs.jproteome.0c01014
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    142
    Proteome Variation with Collagen Yield in Ancient Bone
    Procopio, Noemi; Hopkins, Rachel J. A.; Harvey, Virginia L.; Buckley, Michael
    Journal of Proteome Research (2021), 20 (3), 1754-1769CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Isotope analyses are some of the most common anal. methods applied to ancient bone, aiding the interpretation of past diets and chronol. For this, the evaluation of "collagen yield" (as defined in radiocarbon dating and stable isotope research) is a routine step that allows for the selection of specimens that are deemed adequate for subsequent analyses, with samples contg. less than ~ 1% "collagen yield" normally being used for isotopic anal. but discounted for radiocarbon dating. The aims of this study were to use proteomic methods of MALDI-TOF (matrix assisted laser desorption ionization time-of-fligh mass spectrometry) and LC-ESI-MS/MS (liq. chromatog. electrospray ionization tandem mass spectrometry) to investigate the endogeneity of the dominant proteinaceous biomols. within samples that are typically considered to contain poorly preserved protein. Taking 29 archaeol. samples, we evaluated the proteome variability between different acid-sol. fractions removed prior to protein gelatinization and considered waste as part of the radiocarbon dating process. We then correlated these proteomes against the commonly used "collagen yield" proxy for preservation. We found that these waste fractions contained a significant amt. of both collagenous and noncollagenous proteins (NCPs) but that the abundance of these was not correlated with the acquired "collagen yield". Rather than a depleted protein load as would be expected from a low "collagen yield", the variety of the extd. NCPs was comparable with that commonly obtained from ancient samples and included informative proteins useful for species identification, phylogenetic studies, and potentially even for isotopic analyses, given further method developments. Addnl., we did not observe any correlation between "collagen yield" and peptide mass fingerprint success or between the different fractions taken from the same sample but at different radiocarbon pretreatment stages. Overall, these findings highlight the value in retaining and analyzing sample fractions that are otherwise discarded as waste during the radiocarbon dating process but more importantly, that low "collagen yield" specimens that are often misinterpreted by archaeologists as being devoid of protein can still yield useful mol. sequence-based information.
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  143. 143
    Cleland, T. P.; Sarancha, J. J.; France, C. A. M. Proteomic Profile of Bone “Collagen” Extracted for Stable Isotopes: Implications for Bulk and Single Amino Acid Analyses. Rapid Commun. Mass Spectrom. 2021, 35, e9025  DOI: 10.1002/rcm.9025
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    143
    Proteomic profile of bone "collagen" extracted for stable isotopes: Implications for bulk and single amino acid analyses
    Cleland, Timothy P.; Sarancha, Julianne J.; France, Christine A. M.
    Rapid Communications in Mass Spectrometry (2021), 35 (6), e9025CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Protein studies in archaeol. and paleontol. have been dominated by stable isotope studies to understand diet and trophic levels, but recent applications of proteomic techniques have resulted in a more complete understanding of protein diagenesis than stable isotopes alone. In stable isotope analyses, samples are retained or discarded based on their properties. Proteomics can directly det. what proteins are present within the sample and may be able to allow previously discarded samples to be analyzed. Protein samples that had been previously analyzed for stable isotopes, including those with marginal and poor sample quality, were characterized by liq. chromatog./mass spectrometry using an LTQ Orbitrap Velos mass spectrometer after sepn. on a Dionex Ultimate 3000 LC system. Data were analyzed using MetaMorpheus and custom R scripts. We found a variety of proteins in addn. to collagen, although collagen I was found in the majority of the samples (most samples >80%). We also found a pos. correlation between total deamidation and wt% N, suggesting that deamidation may impact the overall nitrogen signal in bulk analyses. The amino acid profiles of samples, including those of marginal or poor stable isotope quality, reflect the expected collagen I percentages, allowing their use in single amino acid stable isotope analyses. All the samples regardless of quality were found to have high concns. of collagen I, making interpretations of dietary routing based on collagen I reasonably valid. The amino acid profiles on the marginal and poor samples reflect an expected collagen I profile and allow these samples to be recovered for single amino acid analyses.
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    Charlton, S.; Alexander, M.; Collins, M.; Milner, N.; Mellars, P.; O’Connell, T. C.; Stevens, R. E.; Craig, O. E. Finding Britain’s Last Hunter-Gatherers: A New Biomolecular Approach to “Unidentifiable” Bone Fragments Utilising Bone Collagen. J. Archaeol. Sci. 2016, 73, 55– 61,  DOI: 10.1016/j.jas.2016.07.014
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    144
    Finding Britain's last hunter-gatherers: A new biomolecular approach to 'unidentifiable' bone fragments utilising bone collagen
    Charlton, Sophy; Alexander, Michelle; Collins, Matthew; Milner, Nicky; Mellars, Paul; O'Connell, Tamsin C.; Stevens, Rhiannon E.; Craig, Oliver E.
    Journal of Archaeological Science (2016), 73 (), 55-61CODEN: JASCDU; ISSN:1095-9238. (Elsevier Inc.)
    In the last decade, our knowledge of the transition from foraging, fishing, and hunting to agricultural food prodn. has been transformed through the mol. anal. of human remains. In Britain, however, the lack of Late Mesolithic human remains has limited our understanding of this dietary transition. Here, we report the use of a novel strategy to analyze otherwise overlooked material to identify addnl. human remains from this period. ZooMS, a method which uses bone collagen sequences to det. species, was applied to unidentifiable bone fragments from 5th millennium deposits from the Late Mesolithic site of Cnoc Coig (Oronsay, Inner Hebrides) using an innovative new methodol. All samples bar one produced ZooMS results, with 14/20 bone fragments identified as human, and the remainder a mixt. of pig and seal. 70% of bone fragments had sufficient collagen for stable isotope analyses, however none of three human bone fragments analyzed had sufficient endogenous DNA. By conducting AMS dating and stable isotope anal. on this identified collagen, we provide new data that supports the view that the exploitation of marine resources partially overlapped with the earliest agricultural communities in Britain, and thus argues against the idea that forager lifeways in Britain were immediately replaced by agriculture c.4000 cal. BC. Unfortunately, we were unable to explore the genetic relationship between contemporaneous farmers and foragers. However, the more persistent bone protein could be used to identify species, det. date, and assess diet. This novel approach is widely applicable to other early prehistoric sites with fragmentary skeletal material.
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  145. 145
    Fagernäs, Z.; García-Collado, M. I.; Hendy, J.; Hofman, C. A.; Speller, C.; Velsko, I.; Warinner, C. A Unified Protocol for Simultaneous Extraction of DNA and Proteins from Archaeological Dental Calculus. J. Archaeol. Sci. 2020, 118, 105135,  DOI: 10.1016/j.jas.2020.105135
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    Du, J.; Zhu, Z.; Yang, J.; Wang, J.; Jiang, X. A Comparative Study on the Extraction Effects of Common Agents on Collagen-Based Binders in Mural Paintings. Herit. Sci. 2021, 9, 45,  DOI: 10.1186/s40494-021-00519-y
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    146
    A comparative study on the extraction effects of common agents on collagen-based binders in mural paintings
    Du, Jianghao; Zhu, Zhanyun; Yang, Junchang; Wang, Jia; Jiang, Xiaotong
    Heritage Science (2021), 9 (1), 45CODEN: HSECAR; ISSN:2050-7445. (SpringerOpen)
    In this paper, a comparative study was conducted on the extn. effects of six agents for collagen-based mural painting binders. These agents were used to ext. the residual proteins in the non-aged and thermal aged samples. The protein extn. efciencies of diferent extg. agents were quant. detd. by bicinchoninic acid (BCA) method, and then processed by multivariate anal. of variance (MANOVA). The impact of the extn. process on the protein structure was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), UV absorption spectrum (UV) and CD (CD). The results showed that, for both non-aged and aged samples, the extn. efciency of 2 M guanidine hydrochloride (GuHCl) was signifcantly higher than the other fve agents, with less damage to the protein structure during the extn. process.
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  147. 147
    Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal Sample Preparation Method for Proteome Analysis. Nat. Methods 2009, 6, 359– 362,  DOI: 10.1038/nmeth.1322
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    Universal sample preparation method for proteome analysis
    Wisniewski, Jacek R.; Zougman, Alexandre; Nagaraj, Nagarjuna; Mann, Matthias
    Nature Methods (2009), 6 (5), 359-362CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    The authors describe a method, filter-aided sample prepn. (FASP), which combines the advantages of in-gel and in-soln. digestion for mass spectrometry-based proteomics. The authors completely solubilized the proteome in SDS, which the authors then exchanged by urea on a std. filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
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    Fischer, R.; Kessler, B. M. Gel-Aided Sample Preparation (GASP) - A Simplified Method for Gel-Assisted Proteomic Sample Generation from Protein Extracts and Intact Cells. Proteomics 2015, 15, 1224– 1229,  DOI: 10.1002/pmic.201400436
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    148
    Gel-aided sample preparation (GASP)-A simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells
    Fischer, Roman; Kessler, Benedikt M.
    Proteomics (2015), 15 (7), 1224-1229CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We describe a "gel-assisted" proteomic sample prepn. method for MS anal. Solubilized protein exts. or intact cells are copolymd. with acrylamide, facilitating denaturation, redn., quant. cysteine alkylation, and matrix formation. Gel-aided sample prepn. has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein exts. This methodol. is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists.
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  149. 149
    Hendy, J.; Warinner, C.; Bouwman, A.; Collins, M. J.; Fiddyment, S.; Fischer, R.; Hagan, R.; Hofman, C. A.; Holst, M.; Chaves, E. Proteomic Evidence of Dietary Sources in Ancient Dental Calculus. Proc. Biol. Sci. 2018,  DOI: 10.1098/rspb.2018.0977
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    Hughes, C. S.; Foehr, S.; Garfield, D. A.; Furlong, E. E.; Steinmetz, L. M.; Krijgsveld, J. Ultrasensitive Proteome Analysis Using Paramagnetic Bead Technology. Mol. Syst. Biol. 2014, 10, 757,  DOI: 10.15252/msb.20145625
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    150
    Ultrasensitive proteome analysis using paramagnetic bead technology
    Hughes Christopher S; Foehr Sophia; Garfield David A; Furlong Eileen E; Steinmetz Lars M; Krijgsveld Jeroen
    Molecular systems biology (2014), 10 (), 757 ISSN:.
    In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub-microgram amounts of material. These data present a first view of developmental stage-specific proteome dynamics in Drosophila at a single-embryo resolution, permitting characterization of inter-individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.
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  151. 151
    Cleland, T. P. Human Bone Paleoproteomics Utilizing the Single-Pot, Solid-Phase-Enhanced Sample Preparation Method to Maximize Detected Proteins and Reduce Humics. J. Proteome Res. 2018, 17, 3976– 3983,  DOI: 10.1021/acs.jproteome.8b00637
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    151
    Human Bone Paleoproteomics Utilizing the Single-Pot, Solid-Phase-Enhanced Sample Preparation Method to Maximize Detected Proteins and Reduce Humics
    Cleland, Timothy P.
    Journal of Proteome Research (2018), 17 (11), 3976-3983CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Sample prepn. has become an important part of bone proteomics and paleoproteomics and remains one of the major challenges to maximizing the no. of proteins characterized from bone extns. Most paleoproteomic studies have relied on in-soln. digestion with the inclusion of filter-aided sample prepn. (FASP) as effective methods to detect the proteome. However, neither of these are optimal because few proteins have been detected using only in-soln. digestion and the mol. wt. cutoff of FASP may miss remaining fragments of proteins in fossil bone. The recently developed single-pot, solid-phase-enhanced sample prepn. (SP3) overcomes these issues by not relying on mol. wt. while still controlling where the proteins are digested. Here, historical human bones were extd. with either 500 mM tetrasodium EDTA or 400 mM ammonium phosphate dibasic, 200 mM ammonium bicarbonate, 4 M guanidine HCl and digested with the SP3 method. Across all samples, 78 ± 7 (400-200-4) and 79 ± 17 (EDTA) protein accessions were identified, including previously difficult to detect proteins such as osteopontin. SP3 also effectively removed 90% or more of the coextg. humic substances (based on reduced absorbance) from extd. proteins. The utility of SP3 for maximizing the no. of protein detections in historical bones is promising for future paleoproteomic studies.
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  152. 152
    Cleland, T. P. Solid Digestion of Demineralized Bone as a Method To Access Potentially Insoluble Proteins and Post-Translational Modifications. J. Proteome Res. 2018, 17, 536– 542,  DOI: 10.1021/acs.jproteome.7b00670
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    152
    Solid Digestion of Demineralized Bone as a Method To Access Potentially Insoluble Proteins and Post-Translational Modifications
    Cleland, Timothy P.
    Journal of Proteome Research (2018), 17 (1), 536-542CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Bone proteomics is an expanding field for understanding protein changes assocd. with disease as well as characterizing and detecting proteins preserved in fossil bone. Most previous studies used a protocol with demineralization and extn. approach to isolate and characterize proteins from bone. Through near-complete EDTA demineralization, followed by solid digestion of the remaining bone pseudomorph, a total of 92 protein accessions were detected from dog bone. In the EDTA, 14 unique proteins were found, including osteocalcin, an important bone protein. Osteocalcin was not found in the solid digestion samples, demonstrating the importance of examg. the demineralization supernatant. The solid-digestion samples were analyzed both with (11 unique accessions) and without (16 unique accessions) alkylation, resulting in a total of 78 protein accessions. In addn. to the diversity of proteins detected, various post-translational modifications were obsd., including phosphorylation and glycosylation. The solid-digestion approach will allow for characterization of proteins that are insol. and would otherwise be missed by traditional bone protein extn. alone. All data are available at ftp://massive.ucsd.edu/MSV000081399.
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  153. 153
    Jersie-Christensen, R. R.; Sultan, A.; Olsen, J. V. Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity. Methods Mol. Biol. 2016, 1355, 251– 260,  DOI: 10.1007/978-1-4939-3049-4_17
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    153
    Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity
    Jersie-Christensen, Rosa R.; Sultan, Abida; Olsen, Jesper V.
    Methods in Molecular Biology (New York, NY, United States) (2016), 1355 (Phospho-Proteomics), 251-260CODEN: MMBIED; ISSN:1940-6029. (Springer)
    The traditional sample prepn. workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein pptn., redn., alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chem. artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric anal. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, redn., and alkylation are performed in a single step, thus reducing sample loss and increasing reproducibility. Moreover, unlike std. cell lysis procedures the cell harvesting is performed at high temps. (99°C) and without detergents and subsequent need for protein pptn. Phosphopeptides are enriched using TiO2 beads and the orbitrap mass spectrometer is operated in a sensitive mode with higher energy collisional dissocn. (HCD).
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  154. 154
    Cicatiello, P.; Ntasi, G.; Rossi, M.; Marino, G.; Giardina, P.; Birolo, L. Minimally Invasive and Portable Method for the Identification of Proteins in Ancient Paintings. Anal. Chem. 2018, 90, 10128– 10133,  DOI: 10.1021/acs.analchem.8b01718
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    154
    Minimally Invasive and Portable Method for the Identification of Proteins in Ancient Paintings
    Cicatiello, Paola; Ntasi, Georgia; Rossi, Manuela; Marino, Gennaro; Giardina, Paola; Birolo, Leila
    Analytical Chemistry (Washington, DC, United States) (2018), 90 (17), 10128-10133CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A novel method for the anal. of proteinaceous materials present on painted surfaces was developed by taking advantage of the adhesive ability of some fungal proteins which can form a stable and homogeneous layer on flexible transparency sheets able to capture trypsin in a fully active form. The bioactive sheets were able to efficiently digest proteins, present as such, on surfaces of painted tests and historical samples, releasing peptides that can allow an easy and confident identification of the proteinaceous binders by std. bottom-up proteomic approach. By this method there is no need: (1) to transport the artifacts and (2) to remove, even at micro level, a sample from the object. The ingenuity of the method lies in the easily accommodated sampling coupled with a minimal invasiveness.
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  155. 155
    van Doorn, N. L.; Hollund, H.; Collins, M. J. A Novel and Non-Destructive Approach for ZooMS Analysis: Ammonium Bicarbonate Buffer Extraction. Archaeol. Anthropol. Sci. 2011, 3, 281– 289,  DOI: 10.1007/s12520-011-0067-y
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  156. 156
    Fiddyment, S.; Holsinger, B.; Ruzzier, C.; Devine, A.; Binois, A.; Albarella, U.; Fischer, R.; Nichols, E.; Curtis, A.; Cheese, E. Animal Origin of 13th-Century Uterine Vellum Revealed Using Noninvasive Peptide Fingerprinting. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 15066– 15071,  DOI: 10.1073/pnas.1512264112
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    156
    Animal origin of 13th-century uterine vellum revealed using noninvasive peptide fingerprinting
    Fiddyment, Sarah; Holsinger, Bruce; Ruzzier, Chiara; Devine, Alexander; Binois, Annelise; Albarella, Umberto; Fischer, Roman; Nichols, Emma; Curtis, Antoinette; Cheese, Edward; Teasdale, Matthew D.; Checkley-Scott, Caroline; Milner, Stephen J.; Rudy, Kathryn M.; Johnson, Eric J.; Vnoucek, Jiri; Garrison, Mary; McGrory, Simon; Bradley, Daniel G.; Collins, Matthew J.
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (49), 15066-15071CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Tissue-thin parchment made it possible to produce the first pocket Bibles: Thousands were made in the 13th century. The source of this parchment, often called "uterine vellum," has been a long-standing controversy in codicol. Use of the Latin term abortivum in many sources has led some scholars to suggest that the skin of fetal calves or sheep was used. Others have argued that it would not be possible to sustain herds if so many pocket Bibles were produced from fetal skins, arguing instead for unexpected alternatives, such as rabbit. Here, we report a simple and objective technique using std. conservation treatments to identify the animal origin of parchment. The noninvasive method is a variant on zooarchaeol. by mass spectrometry (ZooMS) peptide mass fingerprinting but exts. protein from the parchment surface by using an electrostatic charge generated by gentle rubbing of a PVC eraser on the membrane surface. Using this method, we analyzed 72 pocket Bibles originating in France, England, and Italy and 293 addnl. parchment samples that bracket this period. We found no evidence for the use of unexpected animals; however, we did identify the use of more than one mammal species in a single manuscript, consistent with the local availability of hides. These results suggest that ultrafine vellum does not necessarily derive from the use of abortive or newborn animals with ultrathin hides, but could equally well reflect a prodn. process that allowed the skins of maturing animals of several species to be rendered into vellum of equal quality and fineness.
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  157. 157
    Manfredi, M.; Barberis, E.; Gosetti, F.; Conte, E.; Gatti, G.; Mattu, C.; Robotti, E.; Zilberstein, G.; Koman, I.; Zilberstein, S.; Marengo, E.; Righetti, P. G. Method for Noninvasive Analysis of Proteins and Small Molecules from Ancient Objects. Anal. Chem. 2017, 89, 3310– 3317,  DOI: 10.1021/acs.analchem.6b03722
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    157
    Method for Noninvasive Analysis of Proteins and Small Molecules from Ancient Objects
    Manfredi, Marcello; Barberis, Elettra; Gosetti, Fabio; Conte, Eleonora; Gatti, Giorgio; Mattu, Clara; Robotti, Elisa; Zilberstein, Gleb; Koman, Igor; Zilberstein, Svetlana; Marengo, Emilio; Righetti, Pier Giorgio
    Analytical Chemistry (Washington, DC, United States) (2017), 89 (6), 3310-3317CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Proteins and small mols. from ancient objects and cultural heritage can provide key information and contribute to study the context of objects and artists. However, all present-day protocols and strategies for the anal. of ancient samples are often invasive and require micro sampling. Here, we present a new method for the non-invasive anal. of proteins and small mols.: the technique uses a special ethyl-vinyl acetate film functionalized with strong cation/anion exchange and C8 resins, for interacting with both proteins and small mols. present on the surface of the objects, followed by LC-MS/MS anal. The new method was fully validated for the detn. of both proteins and small mols. on several types of supports, showing excellent anal. performances such as, for example, R2 of the calibration curve of 0.98 and 0.99 for proteins and small mols., low but very repeatable recoveries, particularly adequate for investigations on precious ancient samples that must not be altered by the anal. procedure. ESEM images and LED multispectral imaging confirmed that no damages or alterations occurred onto the support surfaces and no residues were left from the extractive film. Finally the new method was applied for the characterization of the binders of a historical fresco of the XVI century from the Flemish painter Paul Brill, and of a recently discovered fresco from Isidoro Bianchi (XVII century). Moreover, the method was employed for the identification of the colorant used by Pietro Gallo (XIV century) on a wood panel. The method here reported can be easily applied to any other research on ancient precious objects and cultural heritage, since it does not require micro sampling and the proteins/small mols. extn. can be performed directly in situ, leaving the object unchanged and intact.
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  158. 158
    Cleland, T. P.; Vashishth, D. Bone Protein Extraction without Demineralization Using Principles from Hydroxyapatite Chromatography. Anal. Biochem. 2015, 472, 62– 66,  DOI: 10.1016/j.ab.2014.12.006
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    158
    Bone protein extraction without demineralization using principles from hydroxyapatite chromatography
    Cleland, Timothy P.; Vashishth, Deepak
    Analytical Biochemistry (2015), 472 (), 62-66CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    Historically, extn. of bone proteins has relied on the use of demineralization to better retrieve proteins from the extracellular matrix; however, demineralization can be a slow process that restricts subsequent anal. of the samples. Here, the authors developed a novel protein extn. method that does not use demineralization but instead uses a methodol. from hydroxyapatite chromatog. where high concns. of ammonium phosphate and ammonium bicarbonate were used to ext. bone proteins. The authors report that this method has a higher yield than those with previously published small-scale extant bone extns., with and without demineralization. Furthermore, after digestion with trypsin and subsequent HPLC-tandem mass spectrometry (HPLC-MS/MS) anal., the authors were able to detect several extracellular matrix and vascular proteins in addn. to collagen I and osteocalcin. The authors' new method has the potential to isolate proteins within a short period (4 h) and provide information about bone proteins that may be lost during demineralization or using denaturing agents.
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  159. 159
    McGrath, K.; Rowsell, K.; Gates St-Pierre, C.; Tedder, A.; Foody, G.; Roberts, C.; Speller, C.; Collins, M. Identifying Archaeological Bone via Non-Destructive ZooMS and the Materiality of Symbolic Expression: Examples from Iroquoian Bone Points. Sci. Rep. 2019, 9, 11027,  DOI: 10.1038/s41598-019-47299-x
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    159
    Identifying Archaeological Bone via Non-Destructive ZooMS and the Materiality of Symbolic Expression: Examples from Iroquoian Bone Points
    McGrath Krista; Rowsell Keri; Roberts Carolynne; Speller Camilla; Collins Matthew; Rowsell Keri; Gates St-Pierre Christian; Tedder Andrew; Foody George; Speller Camilla; Collins Matthew; Collins Matthew
    Scientific reports (2019), 9 (1), 11027 ISSN:.
    Today, practical, functional and symbolic choices inform the selection of raw materials for worked objects. In cases where we can discern the origin of worked bone, tooth, ivory and antler objects in the past, we assume that similar choices are being made. However, morphological species identification of worked objects is often impossible due to the loss of identifying characteristics during manufacture. Here, we describe a novel non-destructive ZooMS (Zooarchaeology by Mass Spectrometry) method which was applied to bone points from Pre-Contact St. Lawrence Iroquoian village sites in southern Quebec, Canada. The traditional ZooMS technique requires destructive analysis of a sample, which can be problematic when dealing with artefacts. Here we instead extracted proteins from the plastic bags in which the points had been stored. ZooMS analysis revealed hitherto unexpected species, notably black bear (Ursus americanus) and human (Homo sapiens sapiens), used in point manufacture. These surprising results (confirmed through genomic sequencing) highlight the importance of advancing biomolecular research in artefact studies. Furthermore, they unexpectedly and exceptionally allow us to identify and explore the tangible, material traces of the symbolic relationship between bears and humans, central to past and present Iroquoian cosmology and mythology.
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  160. 160
    Ebsen, J. A.; Haase, K.; Larsen, R.; Sommer, D. V. P.; Brandt, L. Ø. Identifying Archaeological Leather - Discussing the Potential of Grain Pattern Analysis and Zooarchaeology by Mass Spectrometry (ZooMS) through a Case Study Involving Medieval Shoe Parts from Denmark. J. Cult. Herit. 2019, 39, 21– 31,  DOI: 10.1016/j.culher.2019.04.008
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    Szpak, P.; Krippner, K.; Richards, M. P. Effects of Sodium Hydroxide Treatment and Ultrafiltration on the Removal of Humic Contaminants from Archaeological Bone. Int. J. Osteoarchaeol. 2017, 27, 1070– 1077,  DOI: 10.1002/oa.2630
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    Oonk, S.; Cappellini, E.; Collins, M. J. Soil Proteomics: An Assessment of Its Potential for Archaeological Site Interpretation. Org. Geochem. 2012, 50, 57– 67,  DOI: 10.1016/j.orggeochem.2012.06.012
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    162
    Soil proteomics: An assessment of its potential for archaeological site interpretation
    Oonk, S.; Cappellini, E.; Collins, M. J.
    Organic Geochemistry (2012), 50 (), 57-67CODEN: ORGEDE; ISSN:0146-6380. (Elsevier Ltd.)
    In this work, two sets of expts. were carried out to assess the potential of soil proteomics for archaeol. site interpretation. First, we examd. the effects of various protein isolation reagents and soil constituents on peptide mass fingerprinting (PMF) of soil-like materials spiked with bovine serum albumin (BSA). In a subsequent case study, we assessed the relative age of soils from an ancient clay floor of a Roman farmhouse using amino acid racemization and then applied MALDI-TOF-MS-MS to detect and identify biomarkers for human occupation. The results from the first expts. indicate that BSA isolation efficiencies are hampered by the presence of clays and reactive org. matter (glucose), whereas these effects are, to some extent, reversed by the presence of humic acids. We also show that specific regions of the BSA mol. (the out most α-helical regions of domain 2 and 3) are more susceptible to isolation than other regions and this suggest that soil proteins can be only partly isolated. Soil-protein interactions were also found to inhibit tryptic cleavage of BSA, resulting in an enhanced specificity of BSA peptides. Our results further stress the importance of multiple or sequential protein isolation protocols with a focus on citrate and hydrofluoric acid. Results from the case study indicate that the ancient floor samples contain degraded and most likely aged proteinaceous matter, probably of keratinaceous origin.
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  163. 163
    Salamon, M.; Tuross, N.; Arensburg, B.; Weiner, S. Relatively Well Preserved DNA Is Present in the Crystal Aggregates of Fossil Bones. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 13783– 13788,  DOI: 10.1073/pnas.0503718102
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    163
    Relatively well preserved DNA is present in the crystal aggregates of fossil bones
    Salamon, Michal; Tuross, Noreen; Arensburg, Baruch; Weiner, Steve
    Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (39), 13783-13788CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    DNA from fossil human bones could provide invaluable information about population migrations, genetic relations between different groups and the spread of diseases. The use of ancient DNA from bones to study the genetics of past populations is, however, very often compromised by the altered and degraded state of preservation of the extd. material. The universally obsd. postmortem degrdn., together with the real possibility of contamination with modern human DNA, makes the acquisition of reliable data, from humans in particular, very difficult. The authors demonstrate that relatively well preserved DNA is occluded within clusters of intergrown bone crystals that are resistant to disaggregation by the strong oxidant NaOCl. The authors obtained reproducible authentic sequences from both modern and ancient animal bones, including humans, from DNA exts. of crystal aggregates. The treatment with NaOCl also minimizes the possibility of modern DNA contamination. The authors thus demonstrate the presence of a privileged niche within fossil bone, which contains DNA in a better state of preservation than the DNA present in the total bone. This counterintuitive approach to extg. relatively well preserved DNA from bones significantly improves the chances of obtaining authentic ancient DNA sequences, esp. from human bones.
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    Zhang, Y.; Fonslow, B. R.; Shan, B.; Baek, M.-C.; Yates, J. R., 3rd. Protein Analysis by Shotgun/Bottom-up Proteomics. Chem. Rev. 2013, 113, 2343– 2394,  DOI: 10.1021/cr3003533
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    Protein Analysis by Shotgun/Bottom-up Proteomics
    Zhang, Yaoyang; Fonslow, Bryan R.; Shan, Bing; Baek, Moon-Chang; Yates, John R.
    Chemical Reviews (Washington, DC, United States) (2013), 113 (4), 2343-2394CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review.
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    Laskay, Ü. A.; Lobas, A. A.; Srzentić, K.; Gorshkov, M. V.; Tsybin, Y. O. Proteome Digestion Specificity Analysis for Rational Design of Extended Bottom-up and Middle-down Proteomics Experiments. J. Proteome Res. 2013, 12, 5558– 5569,  DOI: 10.1021/pr400522h
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    Proteome Digestion Specificity Analysis for Rational Design of Extended Bottom-up and Middle-down Proteomics Experiments
    Laskay, Unige A.; Lobas, Anna A.; Srzentic, Kristina; Gorshkov, Mikhail V.; Tsybin, Yury O.
    Journal of Proteome Research (2013), 12 (12), 5558-5569CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Mass spectrometry (MS)-based bottom-up proteomics (BUP) is currently the method of choice for large-scale identification and characterization of proteins present in complex samples, such as cell lysates, body fluids, or tissues. Tech., BUP relies on MS anal. of complex mixts. of small, <3 kDa, peptides resulting from whole proteome digestion. Because of the extremely high sample complexity, further developments of detection methods and sample prepn. techniques are necessary. In recent years, a no. of alternative approaches such as middle-down proteomics (MDP, addressing up to 15 kDa peptides) and top-down proteomics (TDP, addressing proteins exceeding 15 kDa) have been gaining particular interest. Here we report on the bioinformatics study of both common and less frequently employed digestion procedures for complex protein mixts. specifically targeting the MDP approach. The aim of this study was to maximize the yield of protein structure information from MS data by optimizing peptide size distribution and sequence specificity. We classified peptides into four categories based on mol. wt.: 0.6-3 (classical BUP), 3-7 (extended BUP), 7-15 kDa (MDP), and >15 kDa (TDP). Because of instrumentation-related considerations, we first advocate for the extended BUP approach as the potential near-future improvement of BUP. Therefore, we chose to optimize the no. of unique peptides in the 3-7 kDa range while maximizing the no. of represented proteins. The present study considers human, yeast, and bacterial proteomes. Results of the study can be further used for designing extended BUP or MDP exptl. workflows.
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    Swaney, D. L.; Wenger, C. D.; Coon, J. J. Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics. J. Proteome Res. 2010, 9, 1323– 1329,  DOI: 10.1021/pr900863u
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    166
    Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics
    Swaney, Danielle L.; Wenger, Craig D.; Coon, Joshua J.
    Journal of Proteome Research (2010), 9 (3), 1323-1329CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Large-scale protein sequencing methods rely on enzymic digestion of complex protein mixts. to generate a collection of peptides for mass spectrometric anal. Here the authors examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS2 fragmentation method to peptide precursor, the authors identified 92,095 unique peptides (609,665 total) mapping to 3908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) - 27,822 unique peptides corresponding to 3313 proteins. The addnl. 595 protein identifications were mainly from those at low abundances (i.e., <1000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. The authors demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than tech. replicates, provide a direct route to increase both protein identifications and proteome sequence coverage.
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  167. 167
    Samodova, D.; Hosfield, C. M.; Cramer, C. N.; Giuli, M. V.; Cappellini, E.; Franciosa, G.; Rosenblatt, M. M.; Kelstrup, C. D.; Olsen, J. V. ProAlanase Is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping. Mol. Cell. Proteomics 2020, 19, 2139– 2157,  DOI: 10.1074/mcp.TIR120.002129
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    167
    ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping
    Samodova, Diana; Hosfield, Christopher M.; Cramer, Christian N.; Giuli, Maria V.; Cappellini, Enrico; Franciosa, Giulia; Rosenblatt, Michael M.; Kelstrup, Christian D.; Olsen, Jesper V.
    Molecular & Cellular Proteomics (2020), 19 (12), 2139-2156CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
    Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid compn. of target proteins and the pH of the digestion soln. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymic profiling of a human cell line proteome, histone PTM anal., ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics anal. of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-contg. regions in a nonreduced mAb.
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  168. 168
    Lanigan, L. T.; Mackie, M.; Feine, S.; Hublin, J.-J.; Schmitz, R. W.; Wilcke, A.; Collins, M. J.; Cappellini, E.; Olsen, J. V.; Taurozzi, A. J. Multi-Protease Analysis of Pleistocene Bone Proteomes. J. Proteomics 2020, 228, 103889,  DOI: 10.1016/j.jprot.2020.103889
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    168
    Multi-protease analysis of Pleistocene bone proteomes
    Lanigan, Liam T.; Mackie, Meaghan; Feine, Susanne; Hublin, Jean-Jacques; Schmitz, Ralf W.; Wilcke, Arndt; Collins, Matthew J.; Cappellini, Enrico; Olsen, Jesper V.; Taurozzi, Alberto J.; Welker, Frido
    Journal of Proteomics (2020), 228 (), 103889CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)
    Ancient protein anal. is providing new insights into the evolutionary relationships between hominin fossils across the Pleistocene. Protein identification commonly relies on the proteolysis of a protein ext. using a single protease, trypsin. As with modern proteome studies, alternative or addnl. proteases have the potential to increase both proteome size and protein sequence recovery. This could enhance the recovery of phylogenetic information from ancient proteomes. Here we identify 18 novel hominin bone specimens from the Kleine Feldhofer Grotte using MALDI-TOF MS peptide mass fingerprinting of collagen type I. Next, we use one of these hominin bone specimens and three Late Pleistocene Equidae specimens identified in a similar manner and present a comparison of the bone proteome size and protein sequence recovery obtained after using nanoLC-MS/MS and parallel proteolysis using six different proteases, including trypsin. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also observe that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics. Maximizing proteome and protein sequence recovery of ancient skeletal proteomes is important when analyzing unique hominin fossils. As with modern proteome studies, palaeoproteomic anal. of Pleistocene bone and dentin samples has almost exclusively used trypsin as its only protease, despite the demonstrated advantages of alternative proteases to increase proteome recovery in modern proteome studies. We demonstrate that Pleistocene bone proteomes can be significantly expanded by using addnl. proteases beside trypsin, and that this also improves sequence coverage of individual proteins. The use of several alternative proteases beside trypsin therefore has major benefits to maximize the phylogenetic information retrieved from ancient skeletal proteomes.
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  169. 169
    Calvano, C. D.; Rigante, E. C. L.; Cataldi, T. R. I.; Sabbatini, L. In Situ Hydrogel Extraction with Dual-Enzyme Digestion of Proteinaceous Binders: The Key for Reliable Mass Spectrometry Investigations of Artworks. Anal. Chem. 2020, 92, 10257– 10261,  DOI: 10.1021/acs.analchem.0c01898
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    169
    In Situ Hydrogel Extraction with Dual-Enzyme Digestion of Proteinaceous Binders: the Key for Reliable Mass Spectrometry Investigations of Artworks
    Calvano, Cosima D.; Rigante, Elena C. L.; Cataldi, Tommaso R. I.; Sabbatini, Luigia
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (15), 10257-10261CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A novel strategy based on in situ dual-enzyme digestion of paint layer proteinaceous binders is introduced for faster and more confident identification, resulting in a bottom-up proteomics approach by MALDI-TOF mass spectrometry (MS). In situ sampling/extn. of proteinaceous binders using small pieces of a hydrophilic gel, previously loaded with trypsin and chymotrypsin proteolytic enzymes, was successfully exploited. Along with minimal invasiveness, the synergy of both enzymes was very useful to increase the no. of annotated peptide peaks with their corresponding amino acid sequence by database search and subsequent MALDI-TOF/TOF anal. The protocol was initially aimed at enhancing the identification of egg-based binders and then validated on fresh and aged model pictorial layers; an increased protein coverage was significantly attained regardless of the used painting binders. Optical microscope images and spectrophotocolorimetry anal. evidenced that the painting layers were not damaged or altered because of contact/sampling without leaving hydrogel residues. The proposed protocol was successfully applied on a painted altarpiece "Assumption of the Virgin" dated to the XVI century and on an angel statue of the Nativity crib dated to the XII century, both from Altamura's Cathedral (Apulia, Italy). The occurrence of various protein binders of animal origin was easily and reliably ascertained.
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