Applications in Forensic Proteomics: Protein Identification and Profiling

Proteomics is formally defined as the study of the proteome—the set of all proteins expressed in a cell, tissue, or organism—and its changes with changing environmental conditions. In practice, the term proteomics has come mean the technologies and techniques used to identify and quantitate (in relative or absolute terms) a very large number of proteins (hundreds to thousands) in a single analysis. This chapter introduces the ways in which proteomics analysis can be applied to biological forensics.

Mass-spectrometry-based proteomics approaches and technologies have been employed in numerous areas of the biological sciences and provided many insights into the composition, function, and regulation of biological systems. Despite this long track record in illuminating biological problems, proteomics is just beginning to gain traction in forensic science. Proteomics approaches can identify unknown proteins and organisms, distinguish between various body fluids and tissues, and provide insight into methods of preparation of unknown biological samples. Because forensic proteomics is a nascent field, the concepts, methods, and vocabulary of proteomics may be unfamiliar to forensic scientists. Furthermore, proteomics is a wide and interdisciplinary field that incorporates many different variations of sample preparation, instrumental analysis, and data analysis. This chapter aims to give a brief introduction to the proteomics family of methods, with a focus on principles that will make the other chapters in this volume more accessible. We emphasize the differences between targeted and untargeted proteomics, the process of peptide identification from tandem mass spectrometry data, and the methods of quantitation. Sample preparation is covered in a later chapter.

Proteomic sample preparation techniques rely on dynamic multi-step workflows that are sample classification dependent. As forensic proteomic applications increase in popularity, they will require numerous sample preparation workflows to account for the diverse sample types relevant to this field. As possible examples, a sample may need pretreatment steps to solubilize proteins from a solid surface - clothing, a swab, or a filter. A sample may need to be disrupted or homogenized to liberate proteins trapped in cells. Or a sample may need additional processing steps, such as organic extraction, to isolate proteins from small molecule contaminants which interfere with mass spectrometry measurements. Following these pretreatment steps, proteins will be reduced, denatured, alkylated, and enzymatically digested. The resulting peptide mixture can be desalted by solid phase extraction or fractionated prior to liquid chromatography-mass spectrometry-based proteomic characterization. As a reference for the forensic research community, herein the authors present possible workflows detailing each step of this multi-stage sample type dependent processing to aid with future forensic proteomic applications.

DNA profiling has often been called the “gold standard” for modern forensic testing. The characterization and confirmatory identification of the tissue source of the DNA, however, still presents numerous challenges for forensic serologists. Current antibody- and enzyme activity-based assays used by forensic practitioners for biological stain identification yield only presumptive results. Positive results with non-target body fluids or cross-reactivity with non-human sources have been well documented. Comprehensive proteome mapping and comparative analyses by multidimensional high-performance liquid chromatograph (HPLC) in combination with Q-TOF mass spectrometry have successfully identified and verified a series of protein “biomarker panels” for six body fluids (i.e., peripheral and menstrual blood, vaginal secretions, semen, urine and saliva). Research and development activities have focused on the development of a multiplexed serological assay for the single-pass identification of these body fluids that all have clear forensic relevance. Using an automation platform for front-end sample preparation and a triple quadrupole mass spectrometer coupled to an ultra-high-performance liquid chromatograph (UHPLC) for mass analysis, the resulting workflow was designed to meet the demanding needs of a forensic operational environment. To date, a panel of robust and high-specificity biomarkers for human biological fluid identification has been developed with a run time of only 10 minutes. In conjunction with the automation platform, several hundred samples can be analyzed per week. Rigorous developmental validation studies and testing using casework-type samples have established the accuracy and sensitivity of the assay. The data generated demonstrate the utility of mass spectrometry as a unified platform for both high-sensitivity confirmatory body fluid identification and sample prioritization to optimize downstream genetic analyses.

Body fluid identification is an important adjunct to forensic DNA analysis because it can provide contextual evidence. Protein-based methods are well suited for body fluid detection as protein markers in the three most common forensic body fluids (blood, saliva and semen) are both specific and abundant. There can, however, be inherent difficulties in identifying other forensically important body fluids such as menstrual blood, which is a “mixed” body fluid containing blood, vaginal secretions as well as fragments of endometrial tissue which are specific to menses. While endometrial marker proteins have been identified, their detection can require purification steps that increase cost and time of analysis. An alternative approach using machine learning and based on the relative abundance of both marker and non-marker proteins can be used to build an accurate predictive model. Importantly, a similar approach using regression models can be applied to the deconvolution blood, saliva and semen mixtures as well.

For at least the first three decades since its advent, proteomics has exclusively largely belonged to a clinical, diagnostic, or fundamental biology context. However, the range and the significance of information that proteomes can disclose have led this discipline to be also applied to forensics, ranging from human identification from hair samples, identification of bodily fluids, and microbial forensics to doping investigations. Fingermarks are a relatively new specimen for proteomic studies with any form of proteomic investigation only appearing in 2012 with the analysis of intact peptides and small proteins in situ published by the research group at Sheffield Hallam University. It was not until 2015 that further developments allowed bottom-up proteomics to be also applied directly in situ. While in situ proteomics of fingermarks has many advantages, encompassing simplified sample preparation protocols, speed and the opportunity to perform molecular imaging analyses, this area remains under-investigated. This is probably due to the unique challenges of working with fingermark specimens. The relatively low protein content and the predominantly eccrine origin of fingermarks have been shown to severely impact protein detection at least when the “intact” protein approach is used both in full scan and using a top down approach. In this chapter, advantages, application, challenges and perspective of in situ fingermark proteomics are discussed and compared with classic approaches.

Proteins in biological evidence offer a pathway for human identification when DNA is absent or compromised and can augment existing intact DNA evidence, as collectives of single amino acid substitutions (SAPs) within protein sequences can serve as individual-specific markers. Peptides containing SAPs are known as genetically variant peptides (GVPs). Key to using GVPs in forensics is their link to associated single nucleotide polymorphisms (SNPs) in the corresponding protein-coding DNA. As such, SNP population frequencies can be used to calculate statistics, such as random match probability (RMP), derived from protein evidence, and rules of genetic inheritance can be applied. Proteomic analysis of forensic samples guided by predictions from DNA exomic analysis (i.e., of exons in the genome) can locate these GVPs. Protein-based identification was first demonstrated in 2016 using hair shafts for a cohort of over 60 individuals, producing RMPs up to 1 in 14,000 and ancestry determination. GVPs were shown to persist in archaeological hairs over 250 years old. Subsequent studies have extended GVP capabilities to bone and tooth tissues and shed skin cells. Improved sample preparation and bioinformatics have enabled greater numbers of identified SNPs; a 12,000-fold increase in maximum discriminative power has been achieved even with 100-fold reduction in sample size, from bulk quantities to a single inch of hair. Further, independence of GVP identification from body location-specific protein expression has been demonstrated. Continued development of this technology through common or rare GVP panels and concurrent GVP and mitochondrial DNA analysis provides powerful tools for individual identification and enhanced discriminative power.

Proteomics is becoming ever more popular across a wide variety of disciplines, not only for medical and food-related science but also subject areas such as archaeology, paleontology, and more recently in forensics. As bone is the biological tissue that is the last to decompose, it is the most interesting in the study of proteome decomposition. For ancient bone, the application of proteomic techniques relates to the greater longevity of proteins over DNA, a phenomenon that has been utilized as a means of species identification of ancient bone fragments and the molecular phylogeny of enigmatic extinct species. However, one of the main advantages for forensic scenarios is the greater dynamics of the proteome over the genome. Although our earlier research investigated the decreasing complexity of the bone proteome through deep time (over hundreds of thousands of years), we have recently begun evaluating the changes that occur on more recent, forensic timescales. Published results clearly demonstrate changes in relative abundance of particular proteins that relate to biological age, yet remain informative even on archaeological timescales. However, it is the changes in post-translational modifications that are often utilized to ensure endogeneity in ancient samples, which have recently been found of great use to forensics in their correlation with post-mortem interval estimation. This chapter discusses these and other advances in the application of proteomic methods in forensic science.

Mass spectrometry-based proteomics is a powerful tool for the detection and characterization of microbes of forensic and national security concern. Both targeted and untargeted proteomics methods have been developed for the taxonomic classification of unknown microbial samples. Targeted proteomics assays can be designed for specific microbes of security concern. Untargeted, library-based matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry is now extensively used in the medical field for microbial identification. Several research groups have developed data analysis pipelines for organism identification/taxonomic classification from untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) data, an application which has some overlap with the field of metaproteomics. This chapter reviews these organism identification techniques. Whole genome sequencing/metagenomics is becoming the standard for organism identification/classification. Using examples from published literature, this chapter highlights several examples of how proteomics approaches can provide information that cannot be acquired from DNA sequencing alone, such as distinguishing laboratory-adapted bacteria from closely related wild isolates, and characterizing the growth medium of bacteria and the host cells of virus particles.

Ricin, a ribosomal-inactivating protein toxin produced by the castor plant (Ricinus communis) is considered a biological threat due to its accessibility and low human lethal dose. Method-based mass spectrometry (MS) techniques for the identification of protein toxins such as ricin are needed to enhance analytical processes for data collection on bioforensic samples. MS-based identification of ricin involves comparison of MS measurements between test samples and reference standards in addition to identification of ricin-specific peptides by searching proteome databases.

The National Bioforensic Analysis Center (NBFAC) validated two MS techniques for identification of ricin in bioforensic samples following the ISO 17025 Standard. These methods included: 1) one-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), for separation and identification by molecular weight, followed by in gel trypsin digestion and MALDI TOF/TOF MS analysis and 2) in-solution trypsin digestion and nanoLC-HRMS/MS analysis. Robust method acceptance criteria were established for this ISO 17025 accreditation process to provide confidence in results. Ricin identification is made after all MS data are searched using commercially available proteomics software, compared to data obtained from reference materials analyzed in parallel, and manually reviewed to confirm peptide identifications. The final ricin identification is made when at least one peptide unique to this protein toxin is identified. This validation performed under ISO 17025 standards was completed by multiple technicians over a year-long process, which provided established limits of detection, sensitivity, false positive/negative rates and variability assessments of the methods. ISO 17025 accreditation described in this chapter for these MS techniques for identification provides confidence in the robustness of the processes used during characterization of bioforensic samples containing ricin.

This work was funded under Agreement No. HSHQDC-15-C-00064 awarded to Battelle National Biodefense Institute by the Department of Homeland Security Science and Technology Directorate (DHS S&T) for the management and operation of the National Biodefense Analysis and Countermeasures Center a Federally Funded Research and Development Center. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security or the U.S. Government. The Department of Homeland Security does not endorse any products or commercial services mentioned in this presentation. In no event shall the DHS, BNBI or NBACC have any responsibility or liability for any use, misuse, inability to use, or reliance upon the information contained herein. In addition, no warranty of fitness for a particular purpose, merchantability, accuracy or adequacy is provided regarding the contents of this document

Due to the potential misuse as biothreat agents, ricin and abrin are the focus of surveillance by international and national authorities. Ricin is a protein toxin produced by the castor bean plant (Ricinus communis), whereas abrin is contained in the seeds of Abrus precatorius found in tropical regions. Ricin and abrin belong to the ribosomal inactivating protein class II (RIP II) which possess A- and B-chains. The A-chain mediates the cessation of protein synthesis due to depurination of 28S ribosomal RNA, and the B-chain binds to glycoconjugates of target cells. Fast, sensitive and reliable methods are of great importance for early identification. Advanced mass spectrometric (MS) assays were reported to unambiguously identify ricin or abrin and to detect RIP activity in samples with complex matrices. These assays enable detection and differentiation of ricin or abrin through specific determination of the amino acid sequence of the protein, and active toxins can be monitored from the depurination of a nucleic acid substrate. Because of the inherent susceptibility of MS to matrix effects in the wide range of complex environmental or biological samples investigated in biodefense scenarios, prior purification and enrichment of toxins is most critical for efficient detection. In this review, we describe recent advances in MS-based methods for ricin and abrin detection, with a particular emphasis on affinity extraction protocols.

High molecular weight protein toxins produced by bacteria, e.g. staphylococcal enterotoxins and botulinum neurotoxins, as well as plant toxins such as ricin and abrin, are relevant analytes in different application areas: food safety, public health, civil security and defense sector, and – in case of botulinum neurotoxins – also in pharmaceutics. For their reliable and accurate detection, identification and quantification, reference materials (RMs), in particular certified reference material (CRM), are required. The present article focuses on challenges in the development (production and certification) of such RMs. Firstly, it highlights the role of RMs and CRMs, what they can be used for, the nature of certified properties, metrological traceability, and uncertainty of certified values, as well as commutability of RMs. Secondly, the molecule-specific technical challenges are highlighted using the example of the mentioned toxins. This includes for instance the choice of a suitable purification strategy (recombinant expression and purification versus the purification of toxin from natural sources), the in-depth characterization of the obtained preparations by a comprehensive set of methods including immunochemical assays, mass spectrometry, and functional assays to verify their identity and establish their purity and activity, and finally, suitable approaches for determining reference values of important toxin properties (protein mass concentration in solution, biological activity). The article summarizes ongoing activities in a new European initiative called EuroBioTox, which aims at the production and certification of RMs for selected protein toxins and the establishment of validated procedures for the detection and identification of biological toxins.

The U.S. Federal court system maintains very high standards for admissibility of scientific evidence. In particular, to allow such evidence into court, a judge must be satisfied it is both reliable, in other words, based on a robust, reproducible, and accurate method, and relevant, meaning it adds weight to the prosecution or defense claims in a case. In 1993, the U.S. Supreme Court issued landmark guidelines for judges considering whether or not to admit scientific testimony. The resulting Daubert criteria changed the forensic sciences in a profound way, leading investigators, researchers, and legal experts to question the scientific validity of established forensic methods that had been widely accepted for years. The Daubert criteria also caused a paradigm shift in the development of new forensic methods, forcing researchers to better prepare new techniques for use in an increasingly adversarial environment. In this chapter, we discuss the current state of forensic proteomics in the context of modern forensics. We present the Daubert criteria, discuss seven elements of a defensible method, and provide guidelines for building statistical defensibility of this emerging discipline.