Proteomics for Low Cell Numbers: How to Optimize the Sample Preparation Workflow for Mass Spectrometry AnalysisClick to copy article linkArticle link copied!
- Sara KassemSara KassemDepartment of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Sara Kassem
- Kyra van der PanKyra van der PanDepartment of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Kyra van der Pan
- Anniek L. de JagerAnniek L. de JagerDepartment of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Anniek L. de Jager
- Brigitta A. E. NaberBrigitta A. E. NaberDepartment of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Brigitta A. E. Naber
- Inge F. de LaatInge F. de LaatDepartment of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Inge F. de Laat
- Alesha LouisAlesha LouisDepartment of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Alesha Louis
- Jacques J. M. van Dongen*Jacques J. M. van Dongen*Tel.: +31 71 526 68 71. Email: [email protected]Department of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Jacques J. M. van Dongen
- Cristina TeodosioCristina TeodosioDepartment of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Cristina Teodosio
- Paula DíezPaula DíezDepartment of Immunology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, NetherlandsMore by Paula Díez
Abstract
Nowadays, massive genomics and transcriptomics data can be generated at the single-cell level. However, proteomics in this setting is still a big challenge. Despite the great improvements in sensitivity and performance of mass spectrometry instruments and the better knowledge on sample preparation processing, it is widely acknowledged that multistep proteomics workflows may lead to substantial sample loss, especially when working with paucicellular samples. Still, in clinical fields, frequently limited sample amounts are available for downstream analysis, thereby hampering comprehensive characterization at protein level. To aim at better protein and peptide recoveries, we compare existing and novel approaches in the multistep sample preparation protocols for mass spectrometry studies, from sample collection, cell lysis, protein quantification, and electrophoresis/staining to protein digestion, peptide recovery, and LC-MS/MS instruments. From this critical evaluation, we conclude that the recent innovations and technologies, together with high quality management of samples, make proteomics on paucicellular samples possible, which will have immediate impact for the proteomics community.
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Introduction
Proteomics Workflow for Paucicellular Samples
Cell Sample Collection
Cell Viability Medium
Temperature
Cell Washing
▶ | Tip – Conclusion: To avoid cell death and sample loss: (a) process fresh cell samples as soon as possible; (b) keep the cells in a medium with the right balance between extra molecules (e.g., protease/phosphatase inhibitors), additional protein content, and cell viable environment (e.g., RPMI + 10% FCS); (c) in between steps, keep cells at 4 °C and avoid freeze/thaw cycles; (d) wash cells at 300g for 5 min in cold PBS Na+/K+ at pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) for a maximum of 3 rounds; and (e) proceed immediately with the next step or store cell pellets at −80 °C using snap freezing techniques. |
Cell Lysis
Mechanical Lysis Methods
Nonmechanical Lysis Methods
▶ | Tip – Conclusion: To get efficient lysis from low cell numbers (less than a few thousands of cells): (a) start by thawing your frozen cell pellet (if necessary) in lukewarm water; (b) avoid mechanical-based methods; (c) better to use sonication, enzymes (in a balanced concentration), lysis buffers, or a combination of them; and (d) perform all lysis steps in a single pot to avoid tube-to-tube transfer and related sample losses. |
Protein Quantification
assay name | residue/complex measured (wavelength) | protein detection range (μg/mL) | advantages | incompatibilities | references |
---|---|---|---|---|---|
Colorimetric | |||||
Ultraviolet absorbance | Aromatic residues: Tyr, Trp, Phe (280 nm) | 20–3000 | Easy and quick | Protein mixtures; Some detergents and nucleic acids | (28,40,42) |
Bradford | Dye-protein complex: Coomassie Brilliant Blue G-250 and Arg, His, Trp, Tyr and Phe (595 nm) | 0.2–1500 | Compatible with reducing and chaotropic reagents | Limited sample material; Some detergents | (28,40,42,47,48) |
Biuret | Reduced copper-protein complex (540 nm) | 1000–10 000 | Does not rely on the amino acid composition of the protein | Ammonium salts, sodium phosphate, and glucose | (28,40−42) |
Lowry | Folin-Ciocalteu reagent + Biuret complex (650–750 nm) | 2–1000 | Easy and quick | Reducing agents, strong acids, and EDTA | (28,42−44) |
BCA | BCA + Biuret complex (562 nm) | 0.5–20 (microBCA); 20–2000 (standard BCA) | Compatible with high detergent concentrations and chaotropic agents | Reducing and chelating agents | (28,40−42,45) |
Fluorescent | |||||
Fluorescent assay (e.g., BisANS) | Protein-fluorescent dye complex | 0.02–100 | Optimal for limited sample material; Compatible with chelators, reducing reagents, salts, free nucleotides, solvents, DNA, and protein inhibitors | Large amount of detergents | (28,49) |
Colorimetric Quantification Assays
Biuret
Lowry
BCA
Ultraviolet Absorbance
Bradford
Fluorescent Quantification Assays
▶ | Tip – Conclusion: For protein quantification from low cell numbers, fluorescent-based protein quantification assays offer the greatest sensitivity. |
In-Gel Protein Visualization: Gel Staining Methods
Colorimetric Protein Visualization Methods
Coomassie Brilliant Blue (CBB)
Silver Staining
Fluorescent Protein Visualization Methods
▶ | Tip – Conclusion: To stain limited protein amounts in gels, select optimized silver or fluorescent staining methods as they provide higher sensitivity levels. |
Protein Digestion
▶ | Tip – Conclusion: For protein digestion from paucicellular samples, perform in-solution enzymatic digestion methods in single-pots. Also make sure to adjust the enzyme:protein ratio to allow efficient protein digestion. |
Peptide Recovery
Columns
Beads
▶ | Tip – Conclusion: For peptide recovery from paucicellular samples, opt for bead-based strategies and single-pot processing for a better output and minimized sample losses. |
LC-MS/MS
LC Separation
MS Instrumentation
▶ | Tip – Conclusion: For an improved chromatographic performance: (a) combine long columns (>100 mm) with sufficiently long gradient times (>120 min) and nanoflow rates. (b) For highly reliable and accurate protein identification, MS configurations integrating efficient ionization methods and analyzers with high resolutions and mass accuracies must be employed. |
Others
▶ | Tip – Conclusion: For paucicellular samples use (a) low protein absorption materials made of polypropylene or borosilicate and (b) tubes at low surface area-to-solution volume ratios. |
New Approaches: Advances and Their Applications
technology | cell type description | sample amount (no. of cells, protein amount, sample volume) | no. of identified proteins | mass spectrometer | reference |
---|---|---|---|---|---|
SP3 | Human oocytes | 100 cells | 2154 | Orbitrap Velosf | (91) |
Human oocytes | 1 cell (∼100 ng) | ∼445 | Orbitrap Velosf | (91) | |
Mouse BMDM | 25 000 cell (∼1 μg) | 3152 | Synapt G2-S HDg | (92) | |
HeLa cells | 1 μg | 3300 | |||
Somaless retinal axons from 100 eye explants from Xenopus laevis embryos | ∼2 μg | >1000 | Orbitrap Velosf, Q-Exactivef, Orbitrap Fusionf | (93) | |
Human distal lung resections | 10 mg wet weight | 2412 | Q-Exactive Plusf | (94) | |
Tubules or glomeruli from human kidney | ∼200 cells | >2000 | Q-Exactive Plusf | (95) | |
Microorganism mixture (55% B. subtilis + 35% E. coli + 10% S. cerevisiae) | 1 × 107 CFU (0.3 μg) | 1932 | Q-Exactive HFf | (96) | |
SCoPE-MS | Mouse embryonic stem cells | Single cells | ∼1000 | Orbitrap Elitef | (97) |
Human monocytes/macrophages | 1018 cells | 2700 | Q-Exactivef | (98) | |
AML cell model | Single cells | 1000 | Orbitrap Explorisf | (99) | |
In-StageTip (iST) | S. cerevisiae | 20 μg | 4570 | Q-Exactivef | (9) |
HeLa cells | 20 μg | 9667 | Q-Exactivef | (9) | |
Human peripheral blood | 1 μL (×15) | 313 | Q Exactive HFf | (100) | |
Mouse BMDM | 25 000 cell (∼1 μg) | 2343 | Synapt G2-S HDg | (92) | |
HeLa cells | 1 μg | 3020 | Synapt G2-S HDg | (92) | |
Pediatric urine samples | 0.5 mL (∼130 μg) | 1199 | Q Exactive HFf | (101) | |
Microfluidics | E. colib | 50 ng | 799 | Orbitrap Velosf | (102) |
THP-1 | 1000 cells | 346–911 | Orbitrap Elitef | (103) | |
THP-1 | 100 cells | 275–549 | Orbitrap Elitef | (103) | |
HeLac | 140 cells | ∼3000 | Orbitrap Fusionf | (104) | |
HeLac | Single cells | 670 | Orbitrap Fusionf | (105) | |
HeLac | Single cells | 1056 | Orbitrap Eclipsef | (106) | |
Jurkatd | ∼500 cells | 2500 | Q-Exactive Plusf | (107) | |
Single mouse oocytee | 1 cell | 355 | Orbitrap Elitef | (108) | |
Xenopus laevis embryo | 16-cell embryo | 112 | Orbitrap Fusionf | (109) | |
B and T cellsc | ∼130 cells | 1095 | Orbitrap Explorisf | (110) | |
HeLac | 70/770 cells | 170/620 | Orbitrap Lumosf | (111) | |
MCF10Ac | Single cells | 256 | Orbitrap Fusionf | (112) | |
AMLc | 152 cells | 2558 | Orbitrap Fusionf | (112) | |
Murine cellsc | 72 single cells | 2300 | Orbitrap Fusionf | (113) | |
Laser capture microdissection-coupled | Tomato roots (cortical, epidermal) | 5000–7000 cells | 744–1313 | Orbitrap Elitef, Orbitrap Fusionf | (114) |
Rat brain cortex | 10–18 cells | ∼1000 | Orbitrap Fusionf | (115) | |
Human motor neurons/interneurons | Single cells | ∼1000 | Orbitrap Eclipsef | (106) | |
Prostate tumoral cells (spiked) | 1–5 cells | 164–607 | Orbitrap Fusionf | (116) |
AML, acute myeloid leukemia; BMDM, bone marrow-derived macrophages; CFU, colony-forming unit; SP3, single-pot solid-phase-enhanced sample preparation; SCoPE-MS, single cell proteomics by mass spectrometry.
Capillary zone electrophoresis-tandem mass spectrometry, CZE-MS/MS.
nanoPOTS/autoPOTS.
Digital microfluidics combined with SP3, DMF-SP3.
Nanoliter-scale oil-air-droplet (OAD) chip.
By Thermo Scientific (Waltham, MA, US).
By Waters Corporation (Mildford, MA, US).
SP3 Technology
SCoPE-MS
In-StageTip (iST) Platform
Microfluidics- and Microchip-Based MS Approaches
Laser Capture Microdissection-Coupled Approaches
Conclusion
Acknowledgments
The presented work was funded by the European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme with an ERC Advanced Grant (ERC-2015-AdG 695655, TiMaScan).
Arg | arginine |
Asn | asparagine |
Asp | aspartic acid |
BCA | bicinchoninic acid |
BSA | bovine serum albumin |
CBB | Coomassie Brilliant Blue |
CE | capillary electrophoresis |
CID | collision-induced dissociation |
Cys | cysteine |
DTT | dithiothreitol |
ETD | electron transfer dissociation |
FACS | fluorescence-activated cell sorting |
FASP | filter-aided sample preparation |
FCS | fetal calf serum |
Gly | glycine |
His | histidine |
IAA | iodoacetamide |
iST | in-StageTip digestion |
LCM | laser capture microdissection |
LC | liquid chromatography |
Lys | lysine |
Met | methionine |
MS | mass spectrometry |
Phe | phenylalanine |
Pro | proline |
Q | quadrupole |
SCMS | single-cell mass spectrometric method |
SCoPE-MS | single cell proteomics by mass spectrometry |
SDS | sodium dodecyl sulfate |
SP3 | single-pot solid-phase-enhanced sample preparation |
Trp | tryptophan |
Tyr | tyrosine |
USP3 | Universal Solid-Phase Protein Preparation |
UV | ultraviolet. |
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- 9Kulak, N. A.; Pichler, G.; Paron, I.; Nagaraj, N.; Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 2014, 11 (3), 319– 24, DOI: 10.1038/nmeth.2834Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Smt78%253D&md5=f2feeabae1635d048cf39b9fbb060e8fMinimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cellsKulak, Nils A.; Pichler, Garwin; Paron, Igor; Nagaraj, Nagarjuna; Mann, MatthiasNature Methods (2014), 11 (3), 319-324CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Mass spectrometry (MS)-based proteomics typically employs multistep sample-prepn. workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed vol. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome anal. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy nos. in these cells were highly correlated (R2 = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-no. ests. for 9,667 human proteins and obsd. excellent quant. reproducibility between replicates (R2 = 0.97). The in-StageTip method is straightforward and generally applicable in biol. or clin. applications.
- 10Scheuermann, S.; Schäfer, A.; Langejürgen, J.; Reis, C. A step towards enzyme-free tissue dissociation. Current Directions in Biomedical Engineering 2019, 5 (1), 545, DOI: 10.1515/cdbme-2019-0137Google ScholarThere is no corresponding record for this reference.
- 11Vieira Braga, F. A.; Miragaia, R. J. Tissue Handling and Dissociation for Single-Cell RNA-Seq. Methods Mol. Biol. 2019, 1979, 9– 21, DOI: 10.1007/978-1-4939-9240-9_2Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M%252FpslOqsw%253D%253D&md5=120cc9c7389f5888a72f97a285fa3a1cTissue Handling and Dissociation for Single-Cell RNA-SeqVieira Braga Felipe A; Miragaia Ricardo JMethods in molecular biology (Clifton, N.J.) (2019), 1979 (), 9-21 ISSN:.The starting material for all single-cell protocols is a cell suspension. The particular functions and spatial distribution of immune cells generally make them easy to isolate them from the tissues where they dwell. Here we describe tissue dissociation protocols that have been used to obtain human immune cells from lymphoid and nonlymphoid tissues to be then used as input to single-cell methods. We highlight the main factors that can influence the final quality of single-cell data, namely the stress signatures that can bias its interpretation.
- 12Herzenberg, L. A.; Parks, D.; Sahaf, B.; Perez, O.; Roederer, M.; Herzenberg, L. A. The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford. Clin. Chem. 2002, 48 (10), 1819– 1827, DOI: 10.1093/clinchem/48.10.1819Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XnsV2gsLo%253D&md5=6e4a3175ecbb3bc699d3ad6b073a6e99The history and future of the fluorescence activated cell sorter and flow cytometry: a view from StanfordHerzenberg, Leonard A.; Parks, David; Sahaf, Bita; Perez, Omar; Roederer, Mario; Herzenberg, Leonore A.Clinical Chemistry (Washington, DC, United States) (2002), 48 (10), 1819-1827CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)A review. The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells. Becton Dickinson Immunocytometry Systems introduced the com. machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Lab. and a Becton Dickinson engineering group under Bernie Shoor. Over the years, we have increased the no. of measured FACS dimensions (parameters) and the speed of sorting to where we now simultaneously measure 12 fluorescent colors plus 2 scatter parameters. In this history, I illustrate the great utility of this state-of-the-art instrument, which allows us to simultaneously stain, analyze, and then sort cells from small samples of human blood cells from AIDS patients, infants, stem cell transplant patients, and others. I also illustrate anal. and sorting of multiple subpopulations of lymphocytes by use of 8-12 colors. In addn., I review single cell sorting used to clone and analyze hybridomas and discuss other applications of FACS developed over the past 30 yr, as well as give our ideas on the future of FACS. These ideas are currently being implemented in new programs using the internet for data storage and anal. as well as developing new fluorochromes, e.g., green fluorescent protein and tandem dyes, with applications in such areas as apoptosis, gene expression, cytokine expression, cell biochem., redox regulation, and AIDS. Finally, I describe new FACS methods for measuring activated kinases and phosphatases and redox active enzymes in individual cells simultaneously with cell surface phenotyping. Thus, key functions can be studied in various subsets of cells without the need for prior sorting.
- 13Tung, J. W.; Heydari, K.; Tirouvanziam, R.; Sahaf, B.; Parks, D. R.; Herzenberg, L. A.; Herzenberg, L. A. Modern flow cytometry: a practical approach. Clin Lab Med. 2007, 27 (3), 453– 68, DOI: 10.1016/j.cll.2007.05.001Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2svkt1Oisw%253D%253D&md5=576c5ee1b23035b2bbd7a92d98c74086Modern flow cytometry: a practical approachTung James W; Heydari Kartoosh; Tirouvanziam Rabin; Sahaf Bita; Parks David R; Herzenberg Leonard A; Herzenberg Leonore AClinics in laboratory medicine (2007), 27 (3), 453-68, v ISSN:0272-2712.The demonstration that CD T-cell counts can be used to monitor HIV disease progression opened the way to the first clinical application for fluorescence activated cell sorting (FACS) technology. Modern FACS methodologies such multicolor staining and sorting has opened the way to new and constructive therapeutic and clinical applications. This article outlines approaches in which current users can use to improve the quality of their FACS work without undue effort. FACS technology development and the emergence of new software support for this technology are cooperating in this effort.
- 14Gross, A.; Schoendube, J.; Zimmermann, S.; Steeb, M.; Zengerle, R.; Koltay, P. Technologies for Single-Cell Isolation. Int. J. Mol. Sci. 2015, 16 (8), 16897– 16919, DOI: 10.3390/ijms160816897Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsl2qsrnI&md5=1ddda4b0fe073c4315d6527fd5dc1759Technologies for single-cell isolationGross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, PeterInternational Journal of Molecular Sciences (2015), 16 (8), 16897-16919CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)The handling of single cells is of great importance in applications such as cell line development or single-cell anal., e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell anal. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) resp. Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/diln. (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Addnl., a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstr. to be relevant to the field.
- 15Arntzen, M. Ø.; Thiede, B. ApoptoProteomics, an integrated database for analysis of proteomics data obtained from apoptotic cells. Molecular & cellular proteomics: MCP 2012, 11 (2), M111.010447, DOI: 10.1074/mcp.M111.010447Google ScholarThere is no corresponding record for this reference.
- 16Pawula, M.; Hawthorne, G.; Smith, G. T.; Hill, H. M. Best Practice in Biological Sample Collection, Processing, and Storage for LC-MS in Bioanalysis of Drugs. In Handbook of LC-MS Bioanalysis 2013, 139– 164, DOI: 10.1002/9781118671276.ch13Google ScholarThere is no corresponding record for this reference.
- 17Hodge, K.; Have, S. T.; Hutton, L.; Lamond, A. I. Cleaning up the masses: exclusion lists to reduce contamination with HPLC-MS/MS. J. Proteomics 2013, 88, 92– 103, DOI: 10.1016/j.jprot.2013.02.023Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlsFegu74%253D&md5=0f50a4711381a1ff3fa69c5883a79a73Cleaning up the masses: Exclusion lists to reduce contamination with HPLC-MS/MSHodge, Kelly; Have, Sara Ten; Hutton, Luke; Lamond, Angus I.Journal of Proteomics (2013), 88 (), 92-103CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)Mass spectrometry, in the past five years, has increased in speed, accuracy and use. With the ability of the mass spectrometers to identify increasing nos. of proteins the identification of undesirable peptides (those not from the protein sample) has also increased. Most undesirable contaminants originate in the lab. and come from either the user (e.g. keratin from hair and skin), or from reagents (e.g. trypsin), that are required to prep. samples for anal. We found that a significant amt. of MS instrument time was spent sequencing peptides from abundant contaminant proteins. While completely eliminating non-specific protein contamination is not feasible, it is possible to reduce the sequencing of these contaminants. For example, exclusion lists can provide a list of masses that can be used to instruct the mass spectrometer to 'ignore' the undesired contaminant peptides in the list. We empirically generated be-spoke exclusion lists for several model organisms (Homo sapiens, Caenorhabditis elegans, Saccharomyces cerevisiae and Xenopus laevis), utilizing information from over 500 mass spectrometry runs and cumulative anal. of these data. Here we show that by employing these empirically generated lists, it was possible to reduce the time spent analyzing contaminating peptides in a given sample thereby facilitating more efficient data acquisition and anal. Biol. significanceGiven the current efficacy of the Mass Spectrometry instrumentation, the utilization of data from ~ 500 mass spec runs to generate be-spoke exclusion lists and optimize data acquisition is the significance of this manuscript.T. His article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
- 18Kamlage, B.; Maldonado, S. G.; Bethan, B.; Peter, E.; Schmitz, O.; Liebenberg, V.; Schatz, P. Quality Markers Addressing Preanalytical Variations of Blood and Plasma Processing Identified by Broad and Targeted Metabolite Profiling. Clin. Chem. 2014, 60 (2), 399– 412, DOI: 10.1373/clinchem.2013.211979Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXisVCisro%253D&md5=ab3494bca4c6bc767a7e37872e43b25aQuality markers addressing preanalytical variations of blood and plasma processing identified by broad and targeted metabolite profilingKamlage, Beate; Maldonado, Sandra Gonzalez; Bethan, Bianca; Peter, Erik; Schmitz, Oliver; Liebenberg, Volker; Schatz, PhilippClinical Chemistry (Washington, DC, United States) (2014), 60 (2), 399-412CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Metabolomics is a valuable tool with applications in almost all life science areas. There is an increasing awareness of the essential need for high-quality biospecimens in studies applying omics technologies and biomarker research. Tools to detect effects of both blood and plasma processing are a key for assuring reproducible and credible results. We report on the response of the human plasma metabolome to common preanal. variations in a comprehensive metabolomics anal. to reveal such high-quality markers. Human EDTA blood was subjected to preanal. variations while being processed to plasma: microclotting, prolonged processing times at different temps., hemolysis, and contamination with buffy layer. In a second expt., EDTA plasma was incubated at different temps. for up to 16 h. Samples were subjected to GC-MS and liq. chromatog.-tandem mass spectrometry-based metabolite profiling (MxP Broad Profiling) complemented by targeted methods, i.e., sphingoids (as part of MxP Lipids), MxP Catecholamines, and MxP Eicosanoids. Short-term storage of blood, hemolysis, and short-term storage of noncooled plasma resulted in statistically significant increases of 4% to 19% and decreases of 8% to 12% of the metabolites. Microclotting, contamination of plasma with buffy layer, and short-term storage of cooled plasma were of less impact on the metabolome (0% to 11% of metabolites increased, 0% to 8% decreased). The response of the human plasma metabolome to preanal. variation demands implementation of thorough quality assurance and QC measures to obtain reproducible and credible results from metabolomics studies. Metabolites identified as sensitive to preanalytics can be used to control for sample quality.
- 19Mitchell, B. L.; Yasui, Y.; Li, C. I.; Fitzpatrick, A. L.; Lampe, P. D. Impact of freeze-thaw cycles and storage time on plasma samples used in mass spectrometry based biomarker discovery projects. Cancer Inf. 2005, 1 (1), 98– 104, DOI: 10.1177/117693510500100110Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlaksL3F&md5=793e69972fadb54c92f536dea692735eImpact of freeze-thaw cycles and storage time on plasma samples used in mass spectrometry based biomarker discovery projectsMitchell, Breeana L.; Yasui, Yutaka; Li, Christopher I.; Fitzpatrick, Annette L.; Lampe, Paul D.Cancer Informatics (2005), 1 (1), 98-104CODEN: CIANCJ; ISSN:1176-9351. (Libertas Academica Ltd.)Mass spectrometry approaches to biomarker discovery in human fluids have received a great deal of attention in recent years. While mass spectrometry instrumentation and anal. approaches have been widely investigated, little attention has been paid to how sample handling can impact the plasma proteome and therefore influence biomarker discovery. The authors have investigated the effects of two main aspects of sample handling on MALDI-TOF data: repeated freeze-thaw cycles and the effects of long-term storage of plasma at -70°C. Repeated freeze-thaw cycles resulted in a trend towards increasing changes in peak intensity, particularly after two thaws. However, a 4-yr difference in long-term storage appears to have minimal effect on protein in plasma as no differences in peak no., mass distribution, or coeff. of variation were found between samples. Therefore, limiting freeze/thaw cycles seems more important to maintaining the integrity of the plasma proteome than degrdn. caused by long-term storage at -70°C.
- 20Winter, D.; Dehghani, A.; Steen, H. Optimization of Cell Lysis and Protein Digestion Protocols for Protein Analysis by LC-MS/MS. In Proteomic Profiling: Methods and Protocols; Posch, A., Ed.; Springer: New York, NY, 2015; pp 259– 273.Google ScholarThere is no corresponding record for this reference.
- 21Shehadul Islam, M.; Aryasomayajula, A.; Selvaganapathy, P. R. A Review on Macroscale and Microscale Cell Lysis Methods. Micromachines 2017, 8 (3), 83, DOI: 10.3390/mi8030083Google ScholarThere is no corresponding record for this reference.
- 22Kuhn, A. The Bacterial Cell Wall and Membrane—A Treasure Chest for Antibiotic Targets. In Bacterial Cell Walls and Membranes; Kuhn, A., Ed.; Springer International Publishing: Cham, 2019; pp 1– 5.Google ScholarThere is no corresponding record for this reference.
- 23Rose, G. G. A current interpretation of the anatomy of the mammalian cell. In Mammalian Cell Membranes; Jamieson, G. A., Robinson, D. M., Eds.; Butterworth-Heinemann, 1976; pp 1– 30.Google ScholarThere is no corresponding record for this reference.
- 24Harayama, T.; Riezman, H. Understanding the diversity of membrane lipid composition. Nat. Rev. Mol. Cell Biol. 2018, 19 (5), 281– 296, DOI: 10.1038/nrm.2017.138Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit12mt7g%253D&md5=210fa39fb5e39c8020dc9460e7afcba2Understanding the diversity of membrane lipid compositionHarayama, Takeshi; Riezman, HowardNature Reviews Molecular Cell Biology (2018), 19 (5), 281-296CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)A review. Cellular membranes are formed from a chem. diverse set of lipids present in various amts. and proportions. A high lipid diversity is universal in eukaryotes and is seen from the scale of a membrane leaflet to that of a whole organism, highlighting its importance and suggesting that membrane lipids fulfil many functions. Indeed, alterations of membrane lipid homeostasis are linked to various diseases. While many of their functions remain unknown, interdisciplinary approaches have begun to reveal novel functions of lipids and their interactions. It is begin to understand why even small changes in lipid structures and in compn. can have profound effects on crucial biol. functions.
- 25Raffy, S.; Teissié, J. Control of Lipid Membrane Stability by Cholesterol Content. Biophys. J. 1999, 76 (4), 2072– 2080, DOI: 10.1016/S0006-3495(99)77363-7Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXit1Ojs7o%253D&md5=578da1fff74fa00ce3ec4dc40dfda429Control of lipid membrane stability by cholesterol contentRaffy, Sophie; Teissie, JustinBiophysical Journal (1999), 76 (4), 2072-2080CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Cholesterol has a concn.-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane p.d. modulation induced in vesicles when an external elec. field is applied. The magnitude of this modulation is under the control of the membrane elec. permeability. When brought to a crit. value by the external field, the membrane p.d. induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content.
- 26Goldberg, S. Mechanical/physical methods of cell disruption and tissue homogenization. Methods Mol. Biol. 2008, 424, 3– 22, DOI: 10.1007/978-1-60327-064-9_1Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1c3htFOntg%253D%253D&md5=495e773d762bad1178d1c6c9246d1138Mechanical/physical methods of cell disruption and tissue homogenizationGoldberg StanleyMethods in molecular biology (Clifton, N.J.) (2008), 424 (), 3-22 ISSN:1064-3745.This chapter covers the various methods of mechanical cell disruption and tissue homogenization that are currently commercially available for processing minute samples (<1 ml) to larger production quantities. These mechanical methods of lysing do not introduce chemicals or enzymes to the system. However, the energies needed when using these "harsh" methods can be high and destroy the very proteins being sought. The destruction of cell membranes and walls is effected by subjecting the cells (1) to shearing by liquid flow, (2) to exploding by pressure differences between inside and outside of cell, (3) to collision forces by impact of beads or paddles, or (4) a combination of these forces. Practical suggestions to optimize each method, where to acquire such equipment, and links to reference sources are included.
- 27Feist, P.; Hummon, A. B. Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int. J. Mol. Sci. 2015, 16 (2), 3537– 63, DOI: 10.3390/ijms16023537Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXks1eqtb8%253D&md5=e180d65470db5df37df2fd601e57266cProteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samplesFeist, Peter; Hummon, Amanda B.International Journal of Molecular Sciences (2015), 16 (2), 3537-3563CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biol. samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amts. of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amts. of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amts. of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extn., contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liq. chromatog. and bottom-up mass spectrometry-compatible techniques. Also, a range of biol. specimens, including mammalian tissues and model cell culture systems, are discussed.
- 28Cañas, B.; Piñeiro, C.; Calvo, E.; López-Ferrer, D.; Gallardo, J. M. Trends in sample preparation for classical and second generation proteomics. J. Chromatogr A 2007, 1153 (1–2), 235– 58, DOI: 10.1016/j.chroma.2007.01.045Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXls1Wru78%253D&md5=612a470b0a6f5eb865cc4ff69f397a32Trends in sample preparation for classical and second generation proteomicsCanas, Benito; Pineiro, Carmen; Calvo, Enrique; Lopez-Ferrer, Daniel; Gallardo, Jose ManuelJournal of Chromatography A (2007), 1153 (1-2), 235-258CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)A review. Sample prepn. is a fundamental step in the proteomics workflow. However, it is not easy to find compiled information updating this subject. In this paper, the strategies and protocols for protein extn. and identification, following either classical or second generation proteomics methodologies, are reviewed. Procedures for: tissue disruption, cell lysis, sample pre-fractionation, protein sepn. by 2-DE, protein digestion, mass spectrometry anal., multidimensional peptide sepns. and quantification of protein expression level are described.
- 29DeCaprio, J.; Kohl, T. O. Using Dounce Homogenization to Lyse Cells for Immunoprecipitation. Cold Spring Harb Protoc 2019, 2019 (7), pdb.prot098574, DOI: 10.1101/pdb.prot098574Google ScholarThere is no corresponding record for this reference.
- 30Senichkin, V. V.; Prokhorova, E. A.; Zhivotovsky, B.; Kopeina, G. S. Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells. Cells 2021, 10 (4), 852, DOI: 10.3390/cells10040852Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvF2iu7zP&md5=4a4746ba4ff3b11325669826d294bcc5Simple and efficient protocol for subcellular fractionation of normal and apoptotic cellsSenichkin, Viacheslav V.; Prokhorova, Evgeniia A.; Zhivotovsky, Boris; Kopeina, Gelina S.Cells (2021), 10 (4), 852CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)Subcellular fractionation approaches remain an indispensable tool among a large no. of biochem. methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochem. techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the std. protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the sepn. of nuclear and cytoplasmic contents. The described methodol. is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insol. subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.
- 31Arakawa, T.; Hung, L.; McGinley, M. G.; Rohde, M. F.; Narhi, L. O. Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitor. J. Protein Chem. 1992, 11 (2), 171– 6, DOI: 10.1007/BF01025222Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XksVegur4%253D&md5=ca0cae29c978acb520074799b08303e7Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitorArakawa, Tsutomu; Hung, Lynne; McGinley, Michael G.; Rohde, Michael F.; Narhi, Linda O.Journal of Protein Chemistry (1992), 11 (2), 171-6CODEN: JPCHD2; ISSN:0277-8033.The stabilities of trypsin and soybean trypsin inhibitor in SDS were examd. by SDS-PAGE. Both samples contained several bands, all of which migrated to positions corresponding to the appropriate mol. wt. or less, even when the samples were unheated, suggesting that both the trypsin and trypsin inhibitor are susceptible to SDS-induced denaturation. When they were mixed together prior to addn. of SDS-PAGE sample buffer (1% SDS), a new smearing band appeared which corresponded to a mol. wt. of around 46,000, suggesting that these proteins form a stable complex in SDS. This was confirmed by electroblotting and sequence anal., which indicated that this band contains both the trypsin and inhibitor sequences. At a fixed concn. of the inhibitor, increasing concns. of the trypsin resulted in an increase in the intensity of the complex band. When the mixt. was heated for 10 min in 1% SDS, the complex band disappeared in a temp.-dependent manner. The melting temp. detd. under the exptl. conditions used was about 35°. Similar results were obtained with Bowman-Birk trypsin inhibitor, except that the complex with the above inhibitor had a higher melting temp., around 41°, suggesting that the Bowman-Birk inhibitor/trypsin complex is more stable than the soybean inhibitor/trypsin complex.
- 32Botelho, D.; Wall, M. J.; Vieira, D. B.; Fitzsimmons, S.; Liu, F.; Doucette, A. Top-down and bottom-up proteomics of SDS-containing solutions following mass-based separation. J. Proteome Res. 2010, 9 (6), 2863– 70, DOI: 10.1021/pr900949pGoogle Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlsVSks7s%253D&md5=1433f3715c9377beba0d96a1506ca505Top-Down and Bottom-Up Proteomics of SDS-Containing Solutions Following Mass-Based SeparationBotelho, Diane; Wall, Mark J.; Vieira, Douglas B.; Fitzsimmons, Shayla; Liu, Fang; Doucette, AlanJournal of Proteome Research (2010), 9 (6), 2863-2870CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)SDS has recognized benefits for protein sample prepn., including solubilization and imparting mol. wt. sepn. (e.g., SDS-PAGE). Here, we compare two proteome workflows which incorporate SDS for protein sepn., namely, SDS-PAGE coupled to LC/MS (GeLC MS), along with a soln. sepn. platform, GELFrEE, for intact proteome prefractionation and identification. Despite the clear importance of SDS in these and other proteome anal. workflows, the affect of SDS on an LC/MS proteome expt. has not been quantified. The authors first examd. the influence of SDS on both a bottom-up as well as a top-down (intact protein) MS workflow. Surprisingly, at levels up to 0.01% SDS in the injected sample, reliable MS characterization is obtained. The authors subsequently explored org. pptn. protocols (chloroform/methanol/water and acetone) as a means of lowering SDS, and present a simple modified acetone pptn. protocol which consistently enables MS proteome characterizations from samples initially contg. 2% SDS. With this effective strategy for SDS redn., the GELFrEE MS workflow for bottom-up proteome anal. was characterized relative to GeLC MS. Remarkable agreement in the no. and type of identified proteins was obtained from these two sepn. platforms, validating the use of SDS in soln.-phase proteome anal.
- 33Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S.; Gebler, J. C. Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Anal. Chem. 2003, 75 (21), 6023– 8, DOI: 10.1021/ac0346196Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnsVOitr4%253D&md5=b6307096b452612489304dbd1f13e63fEnzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteinsYu, Ying-Qing; Gilar, Martin; Lee, Peter J.; Bouvier, Edouard S. P.; Gebler, John C.Analytical Chemistry (2003), 75 (21), 6023-6028CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Improved in-soln. tryptic digestion of proteins in terms of speed and peptide coverage was achieved with the aid of a novel acid-labile anionic surfactant (ALS). Unlike SDS, ALS solubilizes proteins without inhibiting trypsin or other common endopeptidases activity. Trypsin activity was evaluated in the presence of various denaturants; little or no decrease in proteolytic activity was obsd. in 0.1-1% ALS solns. (w/v). Sample prepn. prior to mass spectrometry and liq. chromatog. anal. consists of sample acidification. ALS degrades rapidly at low-pH conditions, which eliminates surfactant-caused interference with anal. Described methodol. combines the advantages of protein solubilization, rapid digestion, high peptide coverages, and easy sample prepn. for mass spectrometry and liq. chromatog. analyses.
- 34Wiśniewski, J. R. Filter-Aided Sample Preparation for Proteome Analysis. Methods Mol. Biol. 2018, 1841, 3– 10, DOI: 10.1007/978-1-4939-8695-8_1Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVeqsbzN&md5=ce0d1d579d508bc0d028fc68b0b57befFilter-aided sample preparation for proteome analysisWisniewski, Jacek R.Methods in Molecular Biology (New York, NY, United States) (2018), 1841 (Microbial Proteomics), 3-10CODEN: MMBIED; ISSN:1940-6029. (Springer)A review. Quant. protein extn. and high-yield generation of peptides from biol. samples are the prerequisite for successful bottom-up type proteomic anal. Filter aided sample prepn. (FASP) is a method for processing of SDS-solubilized cells in a proteomic reactor format. In FASP, disposable centrifugal ultrafiltration units allow for detergent depletion, protein digestion, and isolation of peptides released by proteases from undigested material. Consecutive protein digestion with two or three proteases enables generation of peptide fractions with minimal overlap and considerably increases the no. of identifications and protein sequence coverage. FASP is useful for anal. of samples varying in size from a few micrograms to several milligrams of total protein.
- 35Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 2009, 6 (5), 359– 62, DOI: 10.1038/nmeth.1322Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXks12ksb0%253D&md5=f34cb14143462984852497dc1f9ee5c2Universal sample preparation method for proteome analysisWisniewski, Jacek R.; Zougman, Alexandre; Nagaraj, Nagarjuna; Mann, MatthiasNature 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.
- 36Cleland, W. W. Dithiothreitol, a new protective reagent for SH groups. Biochemistry 1964, 3, 480– 2, DOI: 10.1021/bi00892a002Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2cXmvFCqsw%253D%253D&md5=3d97cdc9aaf329681ac68f0b55533469Dithiothreitol, a new protective reagent for SH groupsCleland, W. W.Biochemistry (1964), 3 (4), 480-2CODEN: BICHAW; ISSN:0006-2960.Because of its low oxidn.-redn. potential (-0.33 v. at pH 7), dithiothreitol (and its isomer, dithioerythritol) is capable of maintaining monothiols completely in the reduced state and of reducing disulfides quant. Since this compd. is a highly water-soluble solid with little odor and little tendency to be oxidized directly by air, it should prove much superior to the thiols now used as protective reagents for SH groups.
- 37Deutscher, M. P. Maintaining protein stability. Methods Enzymol. 2009, 463, 121– 7, DOI: 10.1016/S0076-6879(09)63010-XGoogle Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt12jsA%253D%253D&md5=2ae1e8f4f54c4e27f261e7b089e3e0f4Maintaining protein stabilityDeutscher, Murray P.Methods in Enzymology (2009), 463 (Guide to Protein Purification), 121-127CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)Proteins are fragile mols. that often require great care during purifn. to ensure that they remain intact and fully active. Nowadays, many proteins are also purified in small amts. under denaturing conditions by various gel electrophoretic techniques, such that inactive proteins are obtained. But even here, it is usually advantageous to maintain the protein in an intact form. In the case of enzymes, and other proteins with assayable biol. activities, maintenance of activity is generally of prime importance, both for following the protein during purifn. and for subsequent studies of function. This chapter will focus on the major points to keep in mind with regard to maintaining the stability of a protein during purifn. and storage. Various other chapters describe in detail stabilization procedures for specific biol. systems and specific classes of proteins.
- 38Hughes, 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 (10), 757, DOI: 10.15252/msb.20145625Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3ksF2rsA%253D%253D&md5=0a3dce5610fc3f4b8097c6de64ffb9fdUltrasensitive proteome analysis using paramagnetic bead technologyHughes Christopher S; Foehr Sophia; Garfield David A; Furlong Eileen E; Steinmetz Lars M; Krijgsveld JeroenMolecular 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.
- 39Bensaddek, D.; Narayan, V.; Nicolas, A.; Murillo, A. B.; Gartner, A.; Kenyon, C. J.; Lamond, A. I. Micro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level. Proteomics 2016, 16 (3), 381– 392, DOI: 10.1002/pmic.201500264Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjtFCgsw%253D%253D&md5=cd9d5c69316dee6183da674895bf33abMicro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm levelBensaddek, Dalila; Narayan, Vikram; Nicolas, Armel; Brenes Murillo, Alejandro; Gartner, Anton; Kenyon, Cynthia J.; Lamond, Angus I.Proteomics (2016), 16 (3), 381-392CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)Proteomics studies typically analyze proteins at a population level, using exts. prepd. from tens of thousands to millions of cells. The resulting measurements correspond to av. values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the anal. of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin-assocd. factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (), an open access, searchable database resource.
- 40Noble, J. E.; Bailey, M. J. Quantitation of protein. Methods Enzymol. 2009, 463, 73– 95, DOI: 10.1016/S0076-6879(09)63008-1Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt12jsg%253D%253D&md5=496ccb8715492d8e6c0b023259716518Quantitation of proteinNoble, James E.; Bailey, Marc J. A.Methods in Enzymology (2009), 463 (Guide to Protein Purification), 73-95CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)A review. The measurement of protein concn. in an aq. sample is an important assay in biochem. research and development labs for applications ranging from enzymic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly det. the concn. of protein, relative to a std., or using an assigned extinction coeff. Methods are described to provide information on how to analyze protein concn. using UV protein spectroscopy measurements, traditional dye-based absorbance measurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The observation that no single assay dominates the market is due to specific limitations of certain methods that investigators need to consider before selecting the most appropriate assay for their sample. Many of the dye-based assays have unique chem. mechanisms that are prone to interference from chems. prevalent in many biol. buffer prepns. A discussion of which assays are prone to interference and the selection of alternative methods is included.
- 41Sapan, C. V.; Lundblad, R. L.; Price, N. C. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 1999, 29 (2), 99– 108Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXisFWntbk%253D&md5=f36a96d69924bd2c7d6a421b2b6dd2f7Colorimetric protein assay techniquesSapan, Christine V.; Lundblad, Roger L.; Price, Nicholas C.Biotechnology and Applied Biochemistry (1999), 29 (2), 99-108CODEN: BABIEC; ISSN:0885-4513. (Portland Press Ltd.)A review with 145 refs. There has been an increase in the no. of colorimetric assay techniques for the detn. of protein concn. over the past 20 yr. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coeff. of variation/lab.-to-lab. variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most crit. issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a std. for the calibration of the assay; it is crucial that the std. be representative of the sample. If it is not possible to match the std. with the sample from the perspective of protein compn., then it is preferable to use an assay that is not sensitive to the compn. of the protein such as a micro-Kjeldahl technique, quant. amino acid anal. or the biuret assay. In a complex mixt. it might be inappropriate to focus on a general method of protein detn. and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the "gold std." for a given protein, this method needs to be used routinely for calibration.
- 42Goldring, J. P. Protein quantification methods to determine protein concentration prior to electrophoresis. Methods Mol. Biol. 2012, 869, 29– 35, DOI: 10.1007/978-1-61779-821-4_3Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFWrtrbK&md5=c1eb7012ede468cd48aa78300ff03d0aProtein quantification methods to determine protein concentration prior to electrophoresisGoldring, J. P. DeanMethods in Molecular Biology (New York, NY, United States) (2012), 869 (Protein Electrophoresis), 29-35CODEN: MMBIED; ISSN:1064-3745. (Springer)A review. During each step of a protein isolation technique, if enzyme activity is to be detd. and before a protein mixt. is sepd. on a polyacrylamide electrophoresis gel, it is important to det. the concn. of the protein(s) in soln. Measuring protein concn. involves absorbance in the UV range or staining the protein with dyes or copper. This review describes the various protein detn. methods that can be employed to measure protein concn. in soln.
- 43Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193 (1), 265– 75, DOI: 10.1016/S0021-9258(19)52451-6Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG38XhsVyrsw%253D%253D&md5=19d36395fad7dd87661caa3d2b97640dProtein measurement with the Folin phenol reagentLowry, Oliver H.; Rosebrough, Nira J.; Farr, A. Lewis; Randall, Rose J.Journal of Biological Chemistry (1951), 193 (), 265-75CODEN: JBCHA3; ISSN:0021-9258.cf. C.A. 41, 1271h. Proteins were detd. with the Folin phenol reagent after alk. Cu treatment. The method is as sensitive as with Nessler reagent, yet requires no digestion. It is 10-20 times more sensitive than detn. of the ultraviolet absorption at λ = 280 mμ and is more specific. It is several fold more sensitive than the ninhydrin reaction and 100 times more sensitive than the biuret reaction. Two major disadvantages are: the amt. of color varies with different proteins; the color is not strictly proportional to concn. Few substances cause serious interference. Uric acid, guanine, and xanthine react with Folin reagent. The method is useful for following enzyme fractionation, detg. mixed tissue proteins, detn. of very small amts. of protein, or detg. highly dild. protein, or protein in colored solns. or in the presence of N-contg. material.
- 44Stoscheck, C. M. Quantitation of protein. Methods Enzymol. 1990, 182, 50– 68, DOI: 10.1016/0076-6879(90)82008-PGoogle Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXitValsLw%253D&md5=78d0376e0fdafecd901d5e0c9139dc14Quantitation of proteinStoscheck, Christa M.Methods in Enzymology (1990), 182 (Guide Protein Purif.), 50-68CODEN: MENZAU; ISSN:0076-6879.A review with 48 refs. Assays that are easy to perform, require simple instrumentation, and are highly sensitive including methods to conc. samples or to eliminate interfering reagents are discussed.
- 45Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150 (1), 76– 85, DOI: 10.1016/0003-2697(85)90442-7Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlsFKksL0%253D&md5=f4f012331cf4dafa50cdc2d345bade38Measurement of protein using bicinchoninic acidSmith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C.Analytical Biochemistry (1985), 150 (1), 76-85CODEN: ANBCA2; ISSN:0003-2697.A method for the colorimetric detn. of proteins is described which uses bicinchoninic acid to monitor the Cu produced during the biuret reaction. The method (micro and macro) was applied to the detn. of 7 proteins. Bicinchoninic acid forms a 2:1 complex with Cu, resulting in a stable, highly colored chromophore with an absorbance max. at 562 nm. The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concns. When compared to the method of O. H. Lowry et al. (1951), the results reported demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, 1-step anal. and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation assocd. with the Lowry technique.
- 46Desjardins, P.; Hansen, J. B.; Allen, M. Microvolume spectrophotometric and fluorometric determination of protein concentration. Curr. Protoc Protein Sci. 2009, 55, 10, DOI: 10.1002/0471140864.ps0310s55Google ScholarThere is no corresponding record for this reference.
Chapter 3, Unit 3.
- 47Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248– 54, DOI: 10.1016/0003-2697(76)90527-3Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XksVehtrY%253D&md5=43f388a493becc4193c7a49d9de0e1dbA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingBradford, Marion M.Analytical Biochemistry (1976), 72 (1-2), 248-54CODEN: ANBCA2; ISSN:0003-2697.A protein detn. method that involves the binding of coomassie Brilliant Blue G 250 to protein is described. The binding of the dye to protein causes a shift in the absorption max. of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm that is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in ∼ 2 min with good color stability for 1 hr. There is little or no interference from cations such as Na+ or K+ nor from carbohydrates such as sucrose. A small amt. of color is developed in the presence of strongly alk. buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amts. of detergents such as Na dodecyl sulfate, Triton X 100, and commercial glassware detergents. Interference by small amts. of detergent may be eliminated by the use of proper control.
- 48Compton, S. J.; Jones, C. G. Mechanism of dye response and interference in the Bradford protein assay. Anal. Biochem. 1985, 151 (2), 369– 74, DOI: 10.1016/0003-2697(85)90190-3Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XjtVag&md5=3445fe9d97f3abff93c1fb8254360b5bMechanism of dye response and interference in the Bradford protein assayCompton, Steve J.; Jones, Clive G.Analytical Biochemistry (1985), 151 (2), 369-74CODEN: ANBCA2; ISSN:0003-2697.The mechanism of Coomassie Brilliant Blue G-250 dye binding in the spectrophotometric detn. of proteins by the method of M. M. Bradford (1976) was examd. with various polyamino acids, amino acids, and natural products. Bradford Coomassie Brilliant Blue G-250 protein-binding dye exists in 3 forms: cationic, neutral, and anionic. Although the anion is not freely present at the dye reagent pH, it is this form that complexes with protein. Dye binding requires a macromol. form with certain reactive functional groups. Interactions are chiefly with arginine rather than primary amino groups; the other basic and arom. residues give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Assay interference by bases, detergents, and other compds. are explained in terms of their effects upon the equil. between the 3 dye forms.
- 49Datki, Z.; Olah, Z.; Macsai, L.; Pakaski, M.; Galik, B.; Mihaly, G.; Kalman, J. Application of BisANS fluorescent dye for developing a novel protein assay. PLoS One 2019, 14 (4), e0215863 DOI: 10.1371/journal.pone.0215863Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFGrtbbF&md5=46990b80832ffd217f9de984ea972d8eApplication of BisANS fluorescent dye for developing a novel protein assayDatki, Zsolt; Olah, Zita; Macsai, Lilla; Pakaski, Magdolna; Galik, Bence; Mihaly, Gabor; Kalman, JanosPLoS One (2019), 14 (4), e0215863CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)In many biol.- and chem.-related research fields and expts. the quantification of the peptide and/or protein concn. in samples are essential. Every research environment has unique requirements, e.g. metal ions, incubation times, photostability, pH, protease inhibitors, chelators, detergents, etc. A new protein assay may be adequate in different expts. beyond or instead of the well-known std. protocols (e.g. Qubit, Bradford or bicinchoninic acid) in related conceptions. Based on our previous studies, we developed a novel protein assay applying the 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (BisANS) fluorescent dye. This mol. has several advantageous properties related to protein detection: good soly. in water, high photostability at adequate pH, quick interaction kinetics (within seconds) with proteins and no exclusionary sensitivity to the chelator, detergent and inhibitor ingredients. The protocol described in this work is highly sensitive in a large spectrum to detect protein (100-fold dild. samples) concns. (from 0.28 up to more than 100 μg/mL). The BisANS protein assay is valid and applicable for quantification of the amt. of protein in different biol. and/or chem. samples.
- 50Gauci, V. J.; Wright, E. P.; Coorssen, J. R. Quantitative proteomics: assessing the spectrum of in-gel protein detection methods. J. Chem. Biol. 2011, 4 (1), 3– 29, DOI: 10.1007/s12154-010-0043-5Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3Mnhslyiuw%253D%253D&md5=c19fc0af6a75a451793dfd84314ef29dQuantitative proteomics: assessing the spectrum of in-gel protein detection methodsGauci Victoria J; Wright Elise P; Coorssen Jens RJournal of chemical biology (2011), 4 (1), 3-29 ISSN:.Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.
- 51Sundaram, R. K.; Balasubramaniyan, N.; Sundaram, P. Protein stains and applications. Methods Mol. Biol. 2012, 869, 451– 64, DOI: 10.1007/978-1-61779-821-4_39Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFWrt7vJ&md5=84724c904e0cef0afdd35b4c82eccf6aProtein stains and applicationsSundaram, Ranjini K.; Balasubramaniyan, Natarajan; Sundaram, PazhaniMethods in Molecular Biology (New York, NY, United States) (2012), 869 (Protein Electrophoresis), 451-464CODEN: MMBIED; ISSN:1064-3745. (Springer)A review. Staining of proteins sepd. on gels provides the basis for detn. of the crit. properties of these biopolymers, such as their mol. wt. and/or charge. Detection of proteins on gels and blots require stains. These stains vary in sensitivity, ease of use, color, stability, versatility, and specificity. This review discusses different stains and applications with details on how to use the advantages and disadvantages of each stain. It also compiles some important points to be considered in imaging and evaluation. Commonly used colorimetric and fluorescent dyes for general protein staining, and posttranslational modification-specific detection methods are also discussed.
- 52Miller, I.; Crawford, J.; Gianazza, E. Protein stains for proteomic applications: which, when, why?. Proteomics 2006, 6 (20), 5385– 408, DOI: 10.1002/pmic.200600323Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFyms7nE&md5=a02e2a2858005b4a8c978b7c526fd772Protein stains for proteomic applications: Which, when, why?Miller, Ingrid; Crawford, Johanne; Gianazza, ElisabettaProteomics (2006), 6 (20), 5385-5408CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addn. to theor. considerations, examples are provided to illustrate differential staining of specific proteins with different detection methods. This includes a large body of original data on the comparative evaluation of several pre- and post-electrophoresis stains used in parallel on a single specimen, horse serum run in 2-DE (IPG-DALT). A no. of proteins/protein spots are over- or under-revealed with some of the staining procedures.
- 53Patton, W. F. Detection technologies in proteome analysis. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2002, 771 (1–2), 3– 31, DOI: 10.1016/S1570-0232(02)00043-0Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xks1ylsbo%253D&md5=c961db2f7808477fcc146f3cd9eb7171Detection technologies in proteome analysisPatton, Wayne F.Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2002), 771 (1-2), 3-31CODEN: JCBAAI; ISSN:1570-0232. (Elsevier Science B.V.)A review. Common strategies employed for general protein detection include org. dye, silver stain, radiolabeling, reverse stain, fluorescent stain, chemiluminescent stain and mass spectrometry-based approaches. Fluorescence-based protein detection methods have recently surpassed conventional technologies such as colloidal Coomassie blue and silver staining in terms of quant. accuracy, detection sensitivity, and compatibility with modern downstream protein identification and characterization procedures, such as mass spectrometry. Addnl., specific detection methods suitable for revealing protein post-translational modifications have been devised over the years. These include methods for the detection of glycoproteins, phosphoproteins, proteolytic modifications, S-nitrosylation, arginine methylation and ADP-ribosylation. Methods for the detection of a range of reporter enzymes and epitope tags are now available as well, including those for visualizing β-glucuronidase, β-galactosidase, oligohistidine tags and green fluorescent protein. Fluorescence-based and mass spectrometry-based methodologies are just beginning to offer unparalleled new capabilities in the field of proteomics through the performance of multiplexed quant. anal. The primary objective of differential display proteomics is to increase the information content and throughput of proteomics studies through multiplexed anal. Currently, three principal approaches to differential display proteomics are being actively pursued, difference gel electrophoresis (DIGE), multiplexed proteomics (MP) and isotope-coded affinity tagging (ICAT). New multiplexing capabilities should greatly enhance the applicability of the two-dimensional gel electrophoresis technique with respect to addressing fundamental questions related to proteome-wide changes in protein expression and post-translational modification.
- 54Chevalier, F. Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis. Materials 2010, 3 (10), 4784– 4792, DOI: 10.3390/ma3104784Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlKqtLvP&md5=0bc41583c51d4f7558ba0a2c832acdafStandard dyes for total protein staining in gel-based proteomic analysisChevalier, FrancoisMaterials (2010), 3 (), 4784-4792CODEN: MATEG9; ISSN:1996-1944. (Molecular Diversity Preservation International)A review. Staining of two-dimensional gels is a primary concern in proteomic studies using two-dimensional gel electrophoresis with respect to the no. of proteins analyzed, the accuracy of spot quantification and reproducibility. In this review article, the efficiency of the most widely used dyes was investigated. Visible dyes (Coomassie blue and silver nitrate), fluorescent dyes (Sypro Ruby, Deep Purple) and cyanine labeled methods were compared.
- 55Dyballa, N.; Metzger, S. Fast and sensitive coomassie staining in quantitative proteomics. Methods Mol. Biol. 2012, 893, 47– 59, DOI: 10.1007/978-1-61779-885-6_4Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFWrtL7O&md5=5287dbb77e79706f91fc18011815d12bFast and sensitive Coomassie staining in quantitative proteomicsDyballa, Nadine; Metzger, SabineMethods in Molecular Biology (New York, NY, United States) (2012), 893 (Quantitative Methods in Proteomics), 47-59CODEN: MMBIED; ISSN:1064-3745. (Springer)Proteins sepd. by two-dimensional gel electrophoresis can be visualized by in-gel detection using different staining methods. Ideally, the dye should bind non-covalendy to the protein following a linear response curve. Since protein concns. in biol. systems may vary by six or more orders of magnitude, the staining should allow for a detection of very low protein amts. At the same time, satn. effects have to be avoided because they impede normalized quantification. Most proteomics labs. apply Coomassie, silver, or fluorescent stains. Using the colloidal properties of Coomassie dyes, detection limits at the lower nanogram level can meanwhile be achieved. Characteristics like ease of use, low cost, and compatibility with downstream characterization methods such as mass spectrometry, therefore, make colloidal Coomassie staining well suited for the in-gel detection method in quant. proteomics.
- 56Neuhoff, V.; Stamm, R.; Eibl, H. Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis. Electrophoresis 1985, 6 (9), 427– 448, DOI: 10.1002/elps.1150060905Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlslCqtL8%253D&md5=bd40cbdeb05bf36f77b27744133fd7deClear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysisNeuhoff, Volker; Stamm, Reinhard; Eibl, HansjoergElectrophoresis (1985), 6 (9), 427-48CODEN: ELCTDN; ISSN:0173-0835.A systematic anal. of protein staining was carried out in polyacrylamide gels with Coomassie Brilliant Blue (CBB) R-250 and G-250 by using a high resoln. densitometer allowing for quant. measurements during staining and destaining which revealed that none of the published procedures allows quant. measurements. Protein staining with CBB R-250 in MeOH-H2O-HOAc is poor, as is staining with CBB G-250 in trichloroacetic acid or HClO4, the latter 2, however, allowing for a weak background staining. Consequently using the colloidal properties of the CBB dyes, stronger for G-250 than for R-250, it is possible to increase the sensitivity of protein staining to a detection limit of 0.7 ng bovine serum albumin/mm2 gel. In addn., sensitive protein staining on a clear background is possible. Recipes are described for intensified protein staining with CBB G-250 using richloroacetic acid or HClO4 on a clear background. Optimal staining of proteins on a clear background can be performed with phosphoric acid and CBB G-250 in the presence of (NH4)2SO4 since under these conditions the colloidal state of the dye is optimized. Furthermore, conditions are described which allow the stable fixation of the protein-dye complex. Combining the optimized staining conditions with the stable fixation in 20% (NH4)2SO4 allows for stepwise staining for, e.g., detection of weak spots in addn. to intense protein spots. The dependence of different staining procedures on gel thickness, gel concn., and compds. routinely used in polyacrylamide gel electrophoresis is also analyzed. Calibration curves and application of the new procedure to biol. material demonstrate its wide applicability. Convincing arguments for the colloidal properties of the CBB dyes are presented, formulating the rationale for intensified protein staining with CBB dyes in polyacrylamide gels without background staining.
- 57Chevalier, F.; Rofidal, V.; Rossignol, M. Visible and fluorescent staining of two-dimensional gels. Methods Mol. Biol. 2006, 355, 145– 56, DOI: 10.1385/1-59745-227-0:145Google ScholarThere is no corresponding record for this reference.
- 58Harris, L. R.; Churchward, M. A.; Butt, R. H.; Coorssen, J. R. Assessing detection methods for gel-based proteomic analyses. J. Proteome Res. 2007, 6 (4), 1418– 25, DOI: 10.1021/pr0700246Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXivF2isLk%253D&md5=b616525d69ec5378af916cfbaea5d1a9Assessing detection methods for gel-based proteomic analysesHarris, Lauren R.; Churchward, Matthew A.; Butt, R. Hussain; Coorssen, Jens R.Journal of Proteome Research (2007), 6 (4), 1418-1425CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Proteomic analyses using two-dimensional gel electrophoresis (2DE) depend heavily upon the quality of protein stains for sensitive detection. Indeed, detection rather than protein resoln. is likely a current limiting factor in 2DE. The recent development of fluorescent protein stains has dramatically improved the sensitivity of in-gel protein detection and has enabled more accurate protein quantification. Here, we have evaluated the overall quality and relative cost of five com. available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo, and the most commonly used stain, Sypro Ruby (SR). All stains were found to be statistically comparable with regard to no. of protein spots detected, but SR was superior with regard to fluorophore stability (e.g., capacity for repeated use of the stain soln.). Notably, colloidal Coomassie Blue was also found to be comparable to SR when detected using an IR fluorescence imaging system rather than std. densitometry. Thus, depending on available equipment and operating budgets, there are at least two high-sensitivity alternatives to achieve the best currently available in-gel protein detection: Sypro Ruby or Coomassie Blue.
- 59Chevallet, M.; Luche, S.; Rabilloud, T. Silver staining of proteins in polyacrylamide gels. Nat. Protoc. 2006, 1 (4), 1852– 8, DOI: 10.1038/nprot.2006.288Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFagtrnM&md5=f2489e4f3816dff4767bffff65a46bbbSilver staining of proteins in polyacrylamide gelsChevallet, Mireille; Luche, Sylvie; Rabilloud, ThierryNature Protocols (2006), 1 (4), 1852-1858CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Silver staining is used to detect proteins after electrophoretic sepn. on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chems. It is compatible with downstream processing, such as mass spectrometry anal. after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic sepn. Once completed, the stain is stable for several weeks.
- 60Xie, S.; Wong, A. Y. H.; Kwok, R. T. K.; Li, Y.; Su, H.; Lam, J. W. Y.; Chen, S.; Tang, B. Z. Fluorogenic Ag(+) -Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining. Angew. Chem., Int. Ed. 2018, 57 (20), 5750– 5753, DOI: 10.1002/anie.201801653Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnt1elsb4%253D&md5=e7bb70fb87fad8fa66d32366f91d0d15Fluorogenic Ag+-Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver StainingXie, Sheng; Wong, Alex Y. H.; Kwok, Ryan T. K.; Li, Ying; Su, Huifang; Lam, Jacky W. Y.; Chen, Sijie; Tang, Ben ZhongAngewandte Chemie, International Edition (2018), 57 (20), 5750-5753CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Silver staining, which exploits the special bioaffinity and the chromogenic redn. of silver ions, is an indispensable visualization method in biol. It is a most popular method for in-gel protein detection. However, it is limited by run-to-run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) anal.; limitations that are largely attributed to the tricky chromogenic visualization. Herein, the authors reported a novel water-sol. fluorogenic Ag+ probe, the sensing mechanism of which is based on an aggregation-induced emission (AIE) process driven by tetrazolate-Ag+ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.
- 61Magdeldin, S.; Enany, S.; Yoshida, Y.; Xu, B.; Zhang, Y.; Zureena, Z.; Lokamani, I.; Yaoita, E.; Yamamoto, T. Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis. Clin. Proteomics 2014, 11 (1), 16, DOI: 10.1186/1559-0275-11-16Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslShsLnE&md5=5da2864e1ffa73f8e98127a71d5bfa87Basics and recent advances of two dimensional-polyacrylamide gel electrophoresisMagdeldin, Sameh; Enany, Shymaa; Yoshida, Yutaka; Xu, Bo; Zhang, Ying; Zureena, Zam; Lokamani, Ilambarthi; Yaoita, Eishin; Yamamoto, TadashiClinical Proteomics (2014), 11 (), 16/1-16/10, 10 pp.CODEN: CPLRCX; ISSN:1542-6416. (BioMed Central Ltd.)Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional-polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunol. tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its utility still exist. This review discusses the principles of 2-DE as well as both recent methodol. advances and new applications.
- 62Buxbaum, E. Fluorescent Staining of Gels. In Protein Gel Detection and Imaging: Methods and Protocols; Kurien, B. T., Scofield, R. H., Eds.; Springer: New York, NY, 2018; pp 87– 94.Google ScholarThere is no corresponding record for this reference.
- 63Basile, F.; Hauser, N. Rapid online nonenzymatic protein digestion combining microwave heating acid hydrolysis and electrochemical oxidation. Anal. Chem. 2011, 83 (1), 359– 67, DOI: 10.1021/ac1024705Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFaktL7P&md5=0e1196140925b31c095cc0649eee6c75Rapid Online Nonenzymatic Protein Digestion Combining Microwave Heating Acid Hydrolysis and Electrochemical OxidationBasile, Franco; Hauser, NicolasAnalytical Chemistry (Washington, DC, United States) (2011), 83 (1), 359-367CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The authors report an online nonenzymic method for site-specific digestion of proteins to yield peptides that are well suited for collision-induced dissocn. tandem mass spectrometry. The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochem. oxidn. at tryptophan and tyrosine. The combined microwave/electrochem. digestion is reproducible and produces peptides with an av. sequence length of 10 amino acids. This peptide length is similar to the av. peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel nonenzymic digestion method, when analyzed by electrospray ionization mass spectrometry, produce protonated mols. with mostly +1 and +2 charge states. The combination of these two nonenzymic methods overcomes shortcomings with each individual method in that (i) peptides generated by the microwave-hydrolysis method have an av. amino acid length of 16 amino acids and (ii) the electrochem.-cleavage method is unable to reproducibly digest proteins with mol. masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min of digestion time) on a series of std. peptides and proteins as well as an Escherichia coli protein ext.
- 64Inglis, A. S. Cleavage at aspartic acid. Methods Enzymol. 1983, 91, 324– 32, DOI: 10.1016/S0076-6879(83)91030-3Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXktValu7c%253D&md5=1202015554ac259c026b4f656d6ffd95Cleavage at aspartic acidInglis, A. S.Methods in Enzymology (1983), 91 (Enzyme Struct., Pt. I), 324-32CODEN: MENZAU; ISSN:0076-6879.The specific cleavage of peptide bonds after aspartic acid residues in proteins by hydrolysis in dil., pH-2 solns. of HCl and HCO2H for 2 h at 108° under vacuum is discussed. Results for the digestion of several proteins all supported the finding that the majority of aspartyl bonds are cleaved after only 2 h.
- 65Rodríguez, J. C.; Wong, L.; Jennings, P. A. The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides. Protein Expression Purif. 2003, 28 (2), 224– 231, DOI: 10.1016/S1046-5928(02)00700-3Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3s3ivFaitA%253D%253D&md5=d7b2a2a6a8ed6b72c59472f905efe90dThe solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptidesRodriguez Juan Carlos; Wong Lilly; Jennings Patricia AProtein expression and purification (2003), 28 (2), 224-31 ISSN:1046-5928.Abnormal fragmentation during cyanogen bromide polypeptide cleavage rarely occurs, although parallel side reactions are known to typically accompany normal cleavage. We have observed that cyanogen bromide cleavage of highly hydrophobic fusion proteins utilized for production of recombinant peptides results in almost complete abolishment of the expected reaction products when the reaction is carried out in 70% trifluoroacetic acid. On the basis of mass spectrometric analysis of the reaction products, we have identified a number of fragments whose origin can be attributed to incomplete fragmentation of the fusion protein, and to unspecific degradation affecting the carrier protein. Substituting the solvent in the reaction media with 70% formic acid or with a matrix composed of 6M guanidinium hydrochloride in 0.1M HCl, however, was found to alleviate polypeptide cleavage. We have attributed the poor yields of the CNBr cleavage carried out in 70% TFA to the increased hydrophobicity of our particular fusion proteins, and to the poor solubilizing ability of this reaction medium. We propose the utilization of chaotropic agents in the presence of diluted acids as the preferred cyanogen bromide cleavage medium of fusion proteins in order to maximize cleavage efficiency of hydrophobic sequences and to prevent deleterious degradation and structural modifications of the target peptides.
- 66Bornstein, P.; Balian, G. Cleavage at Asn-Gly bonds with hydroxylamine. Methods Enzymol. 1977, 47, 132– 45, DOI: 10.1016/0076-6879(77)47016-2Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXltFShurk%253D&md5=9b08e2b28b03950a93105a57588c543dCleavage at Asn-Gly bonds with hydroxylamineBornstein, Paul; Balian, GaryMethods in Enzymology (1977), 47 (Enzyme Struct., Part E), 132-45CODEN: MENZAU; ISSN:0076-6879.A review with 44 refs. This hydroxylaminolysis can be adapted as a relatively specific means for nonenzymic cleavage of the title bonds in proteins.
- 67Chen, L. C.; Kinoshita, M.; Noda, M.; Ninomiya, S.; Hiraoka, K. Rapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MS. J. Am. Soc. Mass Spectrom. 2015, 26 (7), 1085– 91, DOI: 10.1007/s13361-015-1111-4Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlvVersLw%253D&md5=7ee08b76b1f984260e32bde56d4e7e5bRapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MSChen, Lee Chuin; Kinoshita, Masato; Noda, Masato; Ninomiya, Satoshi; Hiraoka, KenzoJournal of the American Society for Mass Spectrometry (2015), 26 (7), 1085-1091CODEN: JAMSEF; ISSN:1044-0305. (Springer)Recently, we reported a new ESI ion source that could electrospray the super-heated aq. soln. with liq. temp. much higher than the normal b.p. (J. Am. Soc. Mass Spectrom.25, 1862-1869). The boiling of liq. was prevented by pressurizing the ion source to a pressure greater than atm. pressure. The max. operating pressure in our previous prototype was 11 atm, and the highest achievable temp. was 180°C. In this paper, a more compact prototype that can operate up to 27 atm and 250°C liq. temps. is constructed, and reproducible MS acquisition can be extended to electrospray temps. that have never before been tested. Here, we apply this super-heated ESI source to the rapid online protein digestion MS. The sample soln. is rapidly heated when flowing through a heated ESI capillary, and the digestion products are ionized by ESI in situ when the soln. emerges from the tip of the heated capillary. With weak acid such as formic acid as soln., the thermally accelerated digestion (acid hydrolysis) has the selective cleavage at the aspartate (Asp, D) residue sites. The residence time of liq. within the active heating region is about 20 s. The online operation eliminates the need to transfer the sample from the digestion reactor, and the output of the digestive reaction can be monitored and manipulated by the soln. flow rate and heater temp. in a near real-time basis. [Figure not available: see fulltext.].
- 68Giansanti, P.; Tsiatsiani, L.; Low, T. Y.; Heck, A. J. R. Six alternative proteases for mass spectrometry-based proteomics beyond trypsin. Nat. Protoc. 2016, 11 (5), 993– 1006, DOI: 10.1038/nprot.2016.057Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsFaqsLs%253D&md5=e303807cd5bd00ee3dc461b514c8c6abSix alternative proteases for mass spectrometry-based proteomics beyond trypsinGiansanti, Piero; Tsiatsiani, Liana; Low, Teck Yew; Heck, Albert J. R.Nature Protocols (2016), 11 (5), 993-1006CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics expt. Digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in the authors' ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here the authors provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. Data are presented on protease availability and usage including recommendations for reagent prepn. The authors addnl. describe the appropriate MS data anal. methods and the anticipated results in the case of the anal. of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ∼2 d.
- 69Hustoft, H. K.; Malerød, H.; Wilson, S.; Reubsaet, L.; Lundanes, E.; Greibrokk, T. A Critical Review of Trypsin Digestion for LC-MS Based Proteomics. In Integrative Proteomics; Leung, H.-C., Ed.; InTech, 2012; pp 73– 92.Google ScholarThere is no corresponding record for this reference.
- 70Raijmakers, R.; Neerincx, P.; Mohammed, S.; Heck, A. J. R. Cleavage specificities of the brother and sister proteases Lys-C and Lys-N. Chem. Commun. 2009, 46 (46), 8827– 8829, DOI: 10.1039/c0cc02523bGoogle ScholarThere is no corresponding record for this reference.
- 71Gundry, R. L.; White, M. Y.; Murray, C. I.; Kane, L. A.; Fu, Q.; Stanley, B. A.; Van Eyk, J. E. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Current protocols in molecular biology 2009, 25, DOI: 10.1002/0471142727.mb1025s88Google ScholarThere is no corresponding record for this reference.
Chapter 10, Unit 10.
- 72Rappsilber, J.; Ishihama, Y.; Mann, M. Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics. Anal. Chem. 2003, 75 (3), 663– 670, DOI: 10.1021/ac026117iGoogle Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpslOns7w%253D&md5=ae52657518dc8771cdf0868f803e3273Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomicsRappsilber, Juri; Ishihama, Yasushi; Mann, MatthiasAnalytical Chemistry (2003), 75 (3), 663-670CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Proteomics is critically dependent on optimal sample prepn. Particularly, the interface between protein digestion and mass spectrometric anal. has a large influence on the overall quality and sensitivity of the anal. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extn., the procedure has been implemented with com. available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and sepn. material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the prodn. of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 μL/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diam., 0.5 mm length) is 2-4 μg of protein digest, which can be increased by using larger diam. or stacked disks. Five femtomole of tryptic BSA digest could be recovered quant. We have found that the Stage system is well-suited as a universal sample prepn. system for proteomics.
- 73Safarik, I.; Safarikova, M. Magnetic techniques for the isolation and purification of proteins and peptides. BioMagnetic Research and Technology 2004, 2 (1), 7, DOI: 10.1186/1477-044X-2-7Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2sbnvFKjsw%253D%253D&md5=86c8f7ce4fd0ef4c033a02c3b21c9d53Magnetic techniques for the isolation and purification of proteins and peptidesSafarik Ivo; Safarikova MirkaBiomagnetic research and technology (2004), 2 (1), 7 ISSN:1477-044X.Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques.
- 74Waas, M.; Pereckas, M.; Jones Lipinski, R. A.; Ashwood, C.; Gundry, R. L. SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic Analyses. J. Proteome Res. 2019, 18 (4), 1644– 1656, DOI: 10.1021/acs.jproteome.8b00916Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjsFahtb0%253D&md5=a636f6361d78f63262326dfe2b068208SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic AnalysesWaas, Matthew; Pereckas, Michael; Jones Lipinski, Rachel A.; Ashwood, Christopher; Gundry, Rebekah L.Journal of Proteome Research (2019), 18 (4), 1644-1656CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample prepn. steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among labs. To overcome these challenges, the current study systematically evaluated key exptl. parameters regarding the use of carboxylate-modified paramagnetic particles and detd. those that are crit. for max. performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use std. operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concg. the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides and that the approach is compatible with robotic liq. handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will esp. benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.
- 75Kapoor, K. N.; Barry, D. T.; Rees, R. C.; Dodi, I. A.; McArdle, S. E.; Creaser, C. S.; Bonner, P. L. Estimation of peptide concentration by a modified bicinchoninic acid assay. Anal. Biochem. 2009, 393 (1), 138– 140, DOI: 10.1016/j.ab.2009.06.016Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptVersbk%253D&md5=722c1fb8c21d94f44fe20de0a02bcf75Estimation of peptide concentration by a modified bicinchoninic acid assayKapoor, Krishan N.; Barry, Danielle T.; Rees, Robert C.; Anthony Dodi, I.; McArdle, Stephanie E. B.; Creaser, Colin S.; Bonner, Philip L. R.Analytical Biochemistry (2009), 393 (1), 138-140CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)Although biuret based protein assays are theor. applicable to peptide measurement, there is a high level of interpeptide variation, detd. largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95° for 5 min in the presence of 0.1 M NaOH contg. 1% (w/v) SDS, prior to incubation for 30 min at 37° in BCA std. working reagent. This modification to the std. bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estn. of the peptide concn. within an unknown sample.
- 76Sargent, M.; Sage, A.; Wolff, C.; Mussell, C.; Neville, D.; Lord, G.; Saeed, M.; Lad, R.; Godfrey, R.; Hird, S.; Barwick, V. Instrumentation. In Guide to Achieving Reliable Quantitative LC-MS Measurements; Sargent, M., Ed.; RSC Analytical Methods Committee, 2013; pp 5– 11.Google ScholarThere is no corresponding record for this reference.
- 77Shishkova, E.; Hebert, A. S.; Coon, J. J. Now, More Than Ever, Proteomics Needs Better Chromatography. Cell systems 2016, 3 (4), 321– 324, DOI: 10.1016/j.cels.2016.10.007Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFalsrc%253D&md5=0da563a64557756f6d68b7810034c020Now, More Than Ever, Proteomics Needs Better ChromatographyShishkova, Evgenia; Hebert, Alexander S.; Coon, Joshua J.Cell Systems (2016), 3 (4), 321-324CODEN: CSEYA4; ISSN:2405-4712. (Cell Press)From plant research to biomedicine, proteome anal. plays a crit. role in many areas of biol. inquiry. Steady improvement in mass spectrometer (MS) technol. has transformed the speed and depth of proteome anal. Proteomes of simple organisms can now be sequenced to near completion in just over an hour. Comparable coverage of mammalian proteomes, however, still requires hours or even days of anal. Here we ask why current technol. fails to achieve comprehensive and rapid anal. of the more complex mammalian proteomes. We propose that further advancements in MS technol. alone are unlikely to solve this problem and suggest that concomitant improvements in peptide sepn. technol. will be crit.
- 78Chalkley, R. Instrumentation for LC-MS/MS in Proteomics. In LC-MS/MS in Proteomics: Methods and Applications; Cutillas, P. R., Timms, J. F., Eds.; Humana Press: Totowa, NJ, 2010; pp 47– 60.Google ScholarThere is no corresponding record for this reference.
- 79Hsieh, E. J.; Bereman, M. S.; Durand, S.; Valaskovic, G. A.; MacCoss, M. J. Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples. J. Am. Soc. Mass Spectrom. 2013, 24 (1), 148– 153, DOI: 10.1007/s13361-012-0508-6Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVantL0%253D&md5=ffac519618dab9108f941a2acb00911bEffects of Column and Gradient Lengths on Peak Capacity and Peptide Identification in Nanoflow LC-MS/MS of Complex Proteomic SamplesHsieh, Edward J.; Bereman, Michael S.; Durand, Stanley; Valaskovic, Gary A.; MacCoss, Michael J.Journal of the American Society for Mass Spectrometry (2013), 24 (1), 148-153CODEN: JAMSEF; ISSN:1044-0305. (Springer)Reversed-phase liq. chromatog. is the most commonly used sepn. method for shotgun proteomics. Nanoflow chromatog. has emerged as the preferred chromatog. method for its increased sensitivity and sepn. Despite its common use, there are a wide range of parameters and conditions used across research groups. These parameters have an effect on the quality of the chromatog. sepn., which is crit. to maximizing the no. of peptide identifications and minimizing ion suppression. Here we examd. the relationship between column lengths, gradient lengths, peptide identifications, and peptide peak capacity. We found that while longer column and gradient lengths generally increase peptide identifications, the degree of improvement is dependent on both parameters and is diminished at longer column and gradients. Peak capacity, in comparison, showed a more linear increase with column and gradient lengths. We discuss the discrepancy between these two results and some of the considerations that should be taken into account when deciding on the chromatog. conditions for a proteomics expt.
- 80Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. The Column. In Practical HPLC Method Development, 2nd ed.; John Wiley & Sons, 1997; pp 174– 232.Google ScholarThere is no corresponding record for this reference.
- 81Petersson, P.; Frank, A.; Heaton, J.; Euerby, M. R. Maximizing peak capacity and separation speed in liquid chromatography. J. Sep. Sci. 2008, 31 (13), 2346– 57, DOI: 10.1002/jssc.200800064Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXpsFOht7w%253D&md5=5eca042f1610138a5d830bf589bef316Maximizing peak capacity and separation speed in liquid chromatographyPetersson, Patrik; Frank, Andre; Heaton, James; Euerby, Melvin R.Journal of Separation Science (2008), 31 (13), 2346-2357CODEN: JSSCCJ; ISSN:1615-9306. (Wiley-VCH Verlag GmbH & Co. KGaA)The practical effects of gradient time and flow rate on the peak capacities of a range of analytes of differing mol. wts. (MWs) and physico-chem. properties have been evaluated using ultra high pressure LC instrumentation with sub-2 μm and superficially porous particle phases. Optimum peak capacity, in RP gradient LC, for small mols., including typical pharmaceutical drugs and peptides with MWs up to 1300, was demonstrated at a max. flow rate for a given gradient time (i.e. up to 40 min). Flow rates significantly higher than the optimum in the van Deemter plots and also higher than those typically employed by the majority of the chromatographers today are recommended for gradient LC (i.e. up to 1.0 mL/min on 50 - 150 × 2.1 mm 1.7 μm columns). This recommendation is applicable for temps. above 40°, i.e. temps. typically utilized for sepns. employing sub-2 μm particles to reduce column back pressure. Van Deemter and pseudo van Deemter plots were detd. and combined with chromatog. gradient elution theory to explain our unexpected observations. The derived models exhibited good agreement between exptl. and predicted peak capacities (abs. av. error 4%, max. error 12%).
- 82Liu, H.; Finch, J. W.; Lavallee, M. J.; Collamati, R. A.; Benevides, C. C.; Gebler, J. C. Effects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separations. J. Chromatogr A 2007, 1147 (1), 30– 6, DOI: 10.1016/j.chroma.2007.02.016Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjsVSrt7o%253D&md5=ae91b67c04b981f718aa6b6f476ea98aEffects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separationsLiu, Hongji; Finch, Jeffrey W.; Lavallee, Michael J.; Collamati, Robert A.; Benevides, Christopher C.; Gebler, John C.Journal of Chromatography A (2007), 1147 (1), 30-36CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)The effects of the column length, the particle size, the gradient length and the flow rate of a nanoLC system on peptide peak capacity were investigated and compared. Columns packed with 1.7 μm and 3 μm C18 materials into pieces of 75 μm capillary tubing of various lengths were tested with different gradient lengths and flow rates. While increasing the length of a column packed with the 1.7 μm material helped improve peptide peak capacity at the whole range of the tested gradient lengths (24-432 min), little improvement in peak capacity was obsd. with the increase of the length of a column packed with the 3 μm material unless a gradient longer than 50 min was carried out. Up to 30% of peak capacity increase was obsd. when a column's length is doubled, with little redn. in the throughput. In most cases, more than 50% of the increase in peak capacity was obtained with the redn. in the particle size from 3 μm to 1.7 μm. With the same backpressure generated, a shorter 1.7-μm-particle column outperformed a longer column packed with the 3 μm material. In a flow rate range of 100-700 nl/min, increasing the flow rate improved peak capacity for columns packed with 1.7 μm and 3 μm materials.
- 83de Hoffmann, E. Mass Spectrometry. In Kirk-Othmer Encyclopedia of Chemical Technology; Wiley Online Library, 2005; pp 1– 20.Google ScholarThere is no corresponding record for this reference.
- 84Goebel-Stengel, M.; Stengel, A.; Taché, Y.; Reeve, J. R., Jr. The importance of using the optimal plasticware and glassware in studies involving peptides. Anal. Biochem. 2011, 414 (1), 38– 46, DOI: 10.1016/j.ab.2011.02.009Google Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlvVChsrk%253D&md5=e7984ed0611d0477b61d1e575b1e7a2dThe importance of using the optimal plasticware and glassware in studies involving peptidesGoebel-Stengel, Miriam; Stengel, Andreas; Tache, Yvette; Reeve, Joseph R., Jr.Analytical Biochemistry (2011), 414 (1), 38-46CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)The unpredictable nature of peptide binding to surfaces requires optimization of exptl. containers to be used. To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research, the authors tested the recovery of radiolabeled 125I endocrine peptides under different conditions and provide guidelines for detg. the surfaces to use for other peptides. 125I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin-releasing factor, glucagon-like peptide-1 [GLP-1], insulin, leptin, nesfatin-1, and peptide YY), representing a wide spectrum in net charge, size, end group, and modification, were incubated for 48 h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides were incubated with bovine serum albumin (BSA, 1%) with or without subsequent lyophilization. Recovery of 125I-labeled peptides was detd. by gamma counting. Important differences in 125I-labeled peptide binding capacities to various types of surfaces exist. Siliconization decreased, whereas the addn. of BSA improved recovery from surfaces tested. Lyophilizing solns. contg. 125I-labeled peptides and BSA in the tubes best suited for individual peptides rendered more than 89% recovery for all peptides. Ghrelin specifically displaced 125I-ghrelin from borosilicate glass, whereas GLP-1 and Fmoc-arginine did not. Choosing the appropriate exptl. container avoids unpredictable peptide loss that results in inaccurate measurements and false conclusions.
- 85Kraut, A.; Marcellin, M.; Adrait, A.; Kuhn, L.; Louwagie, M.; Kieffer-Jaquinod, S.; Lebert, D.; Masselon, C. D.; Dupuis, A.; Bruley, C.; Jaquinod, M.; Garin, J.; Gallagher-Gambarelli, M. Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples. J. Proteome Res. 2009, 8 (7), 3778– 85, DOI: 10.1021/pr900095uGoogle Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmtlKitbo%253D&md5=3d6dee6b0aedd32de0f31c847c0d5bd7Peptide Storage: Are You Getting the Best Return on Your Investment? Defining Optimal Storage Conditions for Proteomics SamplesKraut, Alexandra; Marcellin, Marlene; Adrait, Annie; Kuhn, Lauriane; Louwagie, Mathilde; Kieffer-Jaquinod, Sylvie; Lebert, Dorothee; Masselon, Christophe D.; Dupuis, Alain; Bruley, Christophe; Jaquinod, Michel; Garin, Jerome; Gallagher-Gambarelli, MaighreadJournal of Proteome Research (2009), 8 (7), 3778-3785CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)To comply with current proteomics guidelines, it is often necessary to analyze the same peptide samples several times. Between analyses, the sample must be stored in such a way as to conserve its intrinsic properties, without losing either peptides or signal intensity. This article describes two studies designed to define the optimal storage conditions for peptide samples between analyses. With the use of a label-free strategy, peptide conservation was compared over a 28-day period in three different recipients: std. plastic tubes, glass tubes, and low-adsorption plastic tubes. The results of this study showed that std. plastic tubes are unsuitable for peptide storage over the period studied. Glass tubes were found to perform better than std. plastic, but optimal peptide recovery was achieved using low-adsorption plastic tubes. The peptides showing poor recovery following storage were mainly hydrophobic in nature. The differences in peptide recovery between glass and low-adsorption plastic tubes were further studied using isotopically labeled proteins. This study allowed accurate comparison of peptide recovery between the two tube types within the same LC-MS run. The results of the label-free study were confirmed. Further, it was possible to demonstrate that peptide recovery in low-adsorption plastic tubes was optimal whatever the peptide concn. stored.
- 86Kristensen, K.; Henriksen, J. R.; Andresen, T. L. Adsorption of cationic peptides to solid surfaces of glass and plastic. PLoS One 2015, 10 (5), e0122419 DOI: 10.1371/journal.pone.0122419Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslylsb3J&md5=9002639cf26eb4da310e6a1a4583385aAdsorption of cationic peptides to solid surfaces of glass and plasticKristensen, Kasper; Henriksen, Jonas R.; Andresen, Thomas L.PLoS One (2015), 10 (5), e0122419/1-e0122419/17CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Cationic membrane-active peptides have been studied for years in the hope of developing them into novel types of therapeutics. In this article, we investigate an effect that might have significant exptl. implications for investigators who wish to study these peptides, namely, that the peptides adsorb to solid surfaces of glass and plastic. Specifically, we use anal. HPLC to systematically quantify the adsorption of the three cationic membrane-active peptides mastoparan X, melittin, and magainin 2 to the walls of commonly used glass and plastic sample containers. Our results show that, at typical exptl. peptide concns., 90% or more of the peptides might be lost from soln. due to rapid adsorption to the walls of the sample containers. Thus, our results emphasize that investigators should always keep these adsorption effects in mind when designing and interpreting expts. on cationic membrane-active peptides. We conclude the article by discussing different strategies for reducing the exptl. impact of these adsorption effects.
- 87Chen, W.-H.; Lee, S.-C.; Sabu, S.; Fang, H.-C.; Chung, S.-C.; Han, C.-C.; Chang, H.-C. Solid-Phase Extraction and Elution on Diamond (SPEED): A Fast and General Platform for Proteome Analysis with Mass Spectrometry. Anal. Chem. 2006, 78 (12), 4228– 4234, DOI: 10.1021/ac052085yGoogle Scholar87https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XksFKksLY%253D&md5=8b128dbe358afa14794c3042d8574a2dSolid-Phase Extraction and Elution on Diamond (SPEED): A Fast and General Platform for Proteome Analysis with Mass SpectrometryChen, Wei-Hao; Lee, Sheng-Chung; Sabu, Sahadevan; Fang, Huei-Chun; Chung, Shu-Chien; Han, Chau-Chung; Chang, Huan-ChengAnalytical Chemistry (2006), 78 (12), 4228-4234CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)This paper presents a new solid-phase extn. and elution platform based on surface-functionalized diamond nanocrystallites. Compared with conventional methods, the platform facilitates purifn. and concn. of intact proteins and their enzymic digests for ensuing SDS-PAGE or matrix-assisted laser desorption/ionization mass spectrometry anal. without prior removal of the adsorbent. One-pot work flow involving redn. of disulfide bonds, protection of free cysteine residues, washing off residual chems., and proteolytic digestion of adsorbed proteins can be performed directly on the particles. The utility and versatility of this protein workup platform were demonstrated with liq. chromatog.-electrospray ionization tandem mass spectrometry in proteome anal. of human urine. The proteome anal. of each urine sample can be completed in 8 h.
- 88DeAngelis, M. M.; Wang, D. G.; Hawkins, T. L. Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 1995, 23 (22), 4742– 4743, DOI: 10.1093/nar/23.22.4742Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXhtVSksLvE&md5=95cde37576ef813b83e0b1e17afb7903Solid-phase reversible immobilization for the isolation of PCR productsDeAngelis, Margaret M.; Wang, David G.; Hawkins, Trevor L.Nucleic Acids Research (1995), 23 (22), 4742-3CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)DNA sequencing directly from PCR products has many advantages over subcloning: the ability to PCR directly from plaques or colonies removes the need for template prepn. and is highly amenable to automation. The main problem with this approach is the subsequent purifn. of the amplified products prior to DNA sequencing. The authors introduce a general method for producing quality DNA sequencing template from PCR products. This procedure is rapid and inexpensive ($0.15 per prep.). The method termed SPRI (solid-phase reversible immobilization) avoids org. extn., filtration and centrifugation steps. The SPRI method employs a carboxy coated magnetic particle manufd. by PerSeptive Diagnostics, Cambride, MA. (cat no #8-4125). The authors discovered that these particles could reversibly bind DNA in the presence of polyethylene glycol (PEG) and salt. This procedure is amenable to automation, is rapid, and yields double-stranded PCR product suitable for DNA sequencing.
- 89Alpert, A. J. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. Journal of Chromatography A 1990, 499, 177– 196, DOI: 10.1016/S0021-9673(00)96972-3Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXkt1Shur8%253D&md5=ae21fa52392f254e07556b563ed5210aHydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compoundsAlpert, Andrew J.Journal of Chromatography (1990), 499 (), 177-96CODEN: JOCRAM; ISSN:0021-9673.When a hydrophilic chromatog. column is eluted with a hydrophobic (mostly org.) mobile phase, retention increases with hydrophilicity of solutes. The term hydrophilic-interaction chromatog. is proposed for this variant of normal-phase chromatog. This mode of chromatog. is of general utility. Mixts. of proteins, peptides, amino acids, oligonucleotides, and carbohydrates are all resolved, with selectivity complementary to those of other modes. Typically, the order of elution is the opposite of that obtained with reversed-phase chromatog. A hydrophilic, neutral packing was developed for use in high-performance hydrophilic-interaction chromatog. Hydrophilic-interaction chromatog. is particularly promising for such troublesome solutes as histones, membrane proteins, and phosphorylated amino acids, and peptides. Hydrophilic-interaction chromatog. fractionations resemble those obtained through partitioning mechanisms. The chromatog. of DNA, in particular, resembles the partitioning obsd. with aq. 2-phase systems based on polyethylene glycol and dextran solns.
- 90Alpert, A. J. Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides. Anal. Chem. 2008, 80 (1), 62– 76, DOI: 10.1021/ac070997pGoogle Scholar90https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlaktLbK&md5=d0055c55a2cdd1c2bcb49d9be0c984d6Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of PhosphopeptidesAlpert, Andrew J.Analytical Chemistry (Washington, DC, United States) (2008), 80 (1), 62-76CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)If an ion-exchange column is eluted with a predominantly org. mobile phase, then solutes can be retained through hydrophilic interaction even if they have the same charge as the stationary phase. This combination is termed electrostatic repulsion-hydrophilic interaction chromatog. (ERLIC). With mixts. of solutes that differ greatly in charge, repulsion effects can be exploited to selectively antagonize the retention of the solutes that normally would be the best retained. This permits the isocratic resoln. of mixts. that normally require gradients, including peptides, amino acids, and nucleotides. ERLIC affords convenient sepns. of highly charged peptides that cannot readily be resolved by other means. In addn., phosphopeptides can be isolated selectively from a tryptic digest.
- 91Virant-Klun, I.; Leicht, S.; Hughes, C.; Krijgsveld, J. Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes. Mol. Cell Proteomics 2016, 15 (8), 2616– 27, DOI: 10.1074/mcp.M115.056887Google Scholar91https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1OktbrF&md5=e9f9a0c5d084fda30f2f466da236a692Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human OocytesVirant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, JeroenMolecular & Cellular Proteomics (2016), 15 (8), 2616-2627CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome compn. and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome anal., resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addn., we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)1 not obsd. in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technol. for proteomic sample prepn. using magnetic beads, we scaled down proteome anal. to single cells. This study demonstrates that an innovative proteomics workflow facilitates anal. of single human oocytes to investigate human oocyte biol. and preimplantation development. The approach presented here paves the way for quant. proteomics in other quantity-limited tissues and cell types. Data assocd. with this study are available via ProteomeXchange with identifier PXD004142.
- 92Sielaff, M.; Kuharev, J.; Bohn, T.; Hahlbrock, J.; Bopp, T.; Tenzer, S.; Distler, U. Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range. J. Proteome Res. 2017, 16 (11), 4060– 4072, DOI: 10.1021/acs.jproteome.7b00433Google Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFCrtb%252FP&md5=5f3d5a92a9186e5aaa0633e6c8121a34Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram RangeSielaff, Malte; Kuharev, Joerg; Bohn, Toszka; Hahlbrock, Jennifer; Bopp, Tobias; Tenzer, Stefan; Distler, UteJournal of Proteome Research (2017), 16 (11), 4060-4072CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Efficient and reproducible sample prepn. is a prerequisite for any robust and sensitive quant. bottom-up proteomics workflow. Here, the authors performed an independent comparison between single-pot solid-phase-enhanced sample prepn. (SP3), filter-aided sample prepn. (FASP), and a com. kit based on the in-StageTip (iST) method. The authors assessed their performance for the processing of proteomic samples in the low μg range using varying amts. of HeLa cell lysate (1-20 μg of total protein). All three workflows showed similar performances for 20 μg of starting material. When handling sample sizes below 10 μg, the no. of identified proteins and peptides as well as the quant. reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low μg range. Even when digesting 1 μg of starting material, both methods still enabled the identification of over 3000 proteins and between 25,000 and 30,000 peptides. On av., the quant. reproducibility between exptl. replicates was slightly higher in case of SP3 (R2 = 0.97 (SP3); R2 = 0.93 (iST)). Applying SP3 toward the characterization of the proteome of FACS-sorted tumor-assocd. macrophages in the B16 tumor model enabled the quantification of 2965 proteins and revealed a "mixed" M1/M2 phenotype.
- 93Cagnetta, R.; Frese, C. K.; Shigeoka, T.; Krijgsveld, J.; Holt, C. E. Rapid Cue-Specific Remodeling of the Nascent Axonal Proteome. Neuron 2018, 99 (1), 29– 46, DOI: 10.1016/j.neuron.2018.06.004Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1Cju7%252FL&md5=90a3fae319528abbf35cf66105636309Rapid Cue-Specific Remodeling of the Nascent Axonal ProteomeCagnetta, Roberta; Frese, Christian K.; Shigeoka, Toshiaki; Krijgsveld, Jeroen; Holt, Christine E.Neuron (2018), 99 (1), 29-46.e4CODEN: NERNET; ISSN:0896-6273. (Cell Press)Axonal protein synthesis and degrdn. are rapidly regulated by extrinsic signals during neural wiring, but the full landscape of proteomic changes remains unknown due to limitations in axon sampling and sensitivity. By combining pulsed stable isotope labeling of amino acids in cell culture with single-pot solid-phase-enhanced sample prepn., we characterized the nascent proteome of isolated retinal axons on an unparalleled rapid timescale (5 min). Our anal. detects 350 basally translated axonal proteins on av., including several linked to neurol. disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) show distinct signatures with more than 100 different nascent protein species up- or downregulated within the first 5 min followed by further dynamic remodeling. Switching repulsion to attraction triggers opposite regulation of a subset of common nascent proteins. Our findings thus reveal the rapid remodeling of the axonal proteomic landscape by extrinsic cues and uncover a logic underlying attraction vs. repulsion.
- 94Åhrman, E.; Hallgren, O.; Malmström, L.; Hedström, U.; Malmström, A.; Bjermer, L.; Zhou, X.-H.; Westergren-Thorsson, G.; Malmström, J. Quantitative proteomic characterization of the lung extracellular matrix in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. J. Proteomics 2018, 189, 23– 33, DOI: 10.1016/j.jprot.2018.02.027Google ScholarThere is no corresponding record for this reference.
- 95Höhne, M.; Frese, C. K.; Grahammer, F.; Dafinger, C.; Ciarimboli, G.; Butt, L.; Binz, J.; Hackl, M. J.; Rahmatollahi, M.; Kann, M.; Schneider, S.; Altintas, M. M.; Schermer, B.; Reinheckel, T.; Göbel, H.; Reiser, J.; Huber, T. B.; Kramann, R.; Seeger-Nukpezah, T.; Liebau, M. C.; Beck, B. B.; Benzing, T.; Beyer, A.; Rinschen, M. M. Single-nephron proteomes connect morphology and function in proteinuric kidney disease. Kidney Int. 2018, 93 (6), 1308– 1319, DOI: 10.1016/j.kint.2017.12.012Google Scholar95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mngt1eqtw%253D%253D&md5=5248f7ac1a0bda6827b81d4698625f28Single-nephron proteomes connect morphology and function in proteinuric kidney diseaseHohne Martin; Schermer Bernhard; Benzing Thomas; Frese Christian K; Grahammer Florian; Dafinger Claudia; Ciarimboli Giuliano; Butt Linus; Binz Julia; Hackl Matthias J; Rahmatollahi Mahdieh; Kann Martin; Schneider Simon; Altintas Mehmet M; Reiser Jochen; Reinheckel Thomas; Gobel Heike; Huber Tobias B; Kramann Rafael; Seeger-Nukpezah Tamina; Liebau Max C; Beck Bodo B; Beyer Andreas; Rinschen Markus MKidney international (2018), 93 (6), 1308-1319 ISSN:.In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale.
- 96Hayoun, K.; Gouveia, D.; Grenga, L.; Pible, O.; Armengaud, J.; Alpha-Bazin, B. Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry. Front. Microbiol. 2019, 10, 1985, DOI: 10.3389/fmicb.2019.01985Google Scholar96https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MnitFKntA%253D%253D&md5=2d438ef71637c66bf9d3ab61035d42b3Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass SpectrometryHayoun Karim; Gouveia Duarte; Grenga Lucia; Pible Olivier; Armengaud Jean; Alpha-Bazin BeatriceFrontiers in microbiology (2019), 10 (), 1985 ISSN:1664-302X.Tandem mass spectrometry-based proteotyping allows characterizing microorganisms in terms of taxonomy and is becoming an important tool for investigating microbial diversity from several ecosystems. Fast and automatable sample preparation for obtaining peptide pools amenable to tandem mass spectrometry is necessary for enabling proteotyping as a high-throughput method. First, the protocol to increase the yield of lysis of several representative bacterial and eukaryotic microorganisms was optimized by using a long and drastic bead-beating setting with 0.1 mm silica beads, 0.1 and 0.5 mm glass beads, in presence of detergents. Then, three different methods to obtain greater digestion yield from these extracts were tested and optimized for improve efficiency and reduce application time: denaturing electrophoresis of proteins and in-gel proteolysis, suspension-trapping filter-based approach (S-Trap) and, solid-phase-enhanced sample preparation named SP3. The latter method outperforms the other two in terms of speed and delivers also more peptides and proteins than with the in-gel proteolysis (2.2 fold for both) and S-trap approaches (1.3 and 1.2 fold, respectively). Thus, SP3 directly improves tandem mass spectrometry proteotyping.
- 97Budnik, B.; Levy, E.; Harmange, G.; Slavov, N. SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. Genome Biol. 2018, 19 (1), 161, DOI: 10.1186/s13059-018-1547-5Google Scholar97https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFKls7nM&md5=bfdeda4d526356689afaa4c6d97a20d5SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiationBudnik, Bogdan; Levy, Ezra; Harmange, Guillaume; Slavov, NikolaiGenome Biology (2018), 19 (), 161/1-161/12CODEN: GNBLFW; ISSN:1474-760X. (BioMed Central Ltd.)Some exciting biol. questions require quantifying thousands of proteins in single cells. To achieve this goal, we develop Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) and validate its ability to identify distinct human cancer cell types based on their proteomes. We use SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem cells. The single-cell proteomes enable us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicates coordinated mRNA and protein covariation, yet many genes exhibit functionally concerted and distinct regulatory patterns at the mRNA and the protein level.
- 98Specht, H.; Emmott, E.; Petelski, A. A.; Huffman, R. G.; Perlman, D. H.; Serra, M.; Kharchenko, P.; Koller, A.; Slavov, N. Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2. Genome Biol. 2021, 22 (1), 50, DOI: 10.1186/s13059-021-02267-5Google Scholar98https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXls1Kmt78%253D&md5=118dd504e7879d91079362cf8a14675fSingle-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2Specht, Harrison; Emmott, Edward; Petelski, Aleksandra A.; Huffman, R. Gray; Perlman, David H.; Serra, Marco; Kharchenko, Peter; Koller, Antonius; Slavov, NikolaiGenome Biology (2021), 22 (1), 50CODEN: GNBLFW; ISSN:1474-760X. (BioMed Central Ltd.)Abstr.: Background: Macrophages are innate immune cells with diverse functional and mol. phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quant. single-cell protein anal. Results: To overcome this limitation, we develop SCoPE2, which substantially increases quant. accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample prepn. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10x Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Conclusions: Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodol. lays the foundation for automated and quant. single-cell anal. of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.
- 99Schoof, E. M.; Furtwängler, B.; Üresin, N.; Rapin, N.; Savickas, S.; Gentil, C.; Lechman, E.; Auf dem Keller, U.; Dick, J. E.; Porse, B. T. Quantitative Single-Cell Proteomics as a Tool to Characterize Cellular Hierarchies. Nat. Commun. 2021, 12, 3341, DOI: 10.1038/s41467-021-23667-yGoogle Scholar99https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVOqtrjP&md5=4143f4cbf8ea4d84e4b372b5bcaae38bQuantitative single-cell proteomics as a tool to characterize cellular hierarchiesSchoof, Erwin M.; Furtwangler, Benjamin; Uresin, Nil; Rapin, Nicolas; Savickas, Simonas; Gentil, Coline; Lechman, Eric; Keller, Ulrich auf dem; Dick, John E.; Porse, Bo T.Nature Communications (2021), 12 (1), 3341CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Large-scale single-cell analyses are of fundamental importance in order to capture biol. heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked exptl. and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying ∼1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we develop a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream anal. The approach presented here lays a foundation for implementing global single-cell proteomics studies across the world.
- 100Geyer, P. E.; Kulak, N. A.; Pichler, G.; Holdt, L. M.; Teupser, D.; Mann, M. Plasma Proteome Profiling to Assess Human Health and Disease. Cell Syst 2016, 2 (3), 185– 95, DOI: 10.1016/j.cels.2016.02.015Google Scholar100https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFKks7s%253D&md5=a7279118db45c785d494798a59a7cbefPlasma Proteome Profiling to Assess Human Health and DiseaseGeyer, Philipp E.; Kulak, Nils A.; Pichler, Garwin; Holdt, Lesca M.; Teupser, Daniel; Mann, MatthiasCell Systems (2016), 2 (3), 185-195CODEN: CSEYA4; ISSN:2405-4712. (Cell Press)Proteins in the circulatory system mirror an individual's physiol. In daily clin. practice, protein levels are generally detd. using single-protein immunoassays. High-throughput, quant. anal. using mass-spectrometry-based proteomics of blood, plasma, and serum would be advantageous but is challenging because of the high dynamic range of protein abundances. Here, we introduce a rapid and robust "plasma proteome profiling" pipeline. This single-run shotgun proteomic workflow does not require protein depletion and enables quant. anal. of hundreds of plasma proteomes from 1μl single finger pricks with 20 min gradients. The apolipoprotein family, inflammatory markers such as C-reactive protein, gender-related proteins, and >40 FDA-approved biomarkers are reproducibly quantified (CV <20% with label-free quantification). Furthermore, we functionally interpret a 1,000-protein, quant. plasma proteome obtained by simple peptide pre-fractionation. Plasma proteome profiling delivers an informative portrait of a person's health state, and we envision its large-scale use in biomedicine.
- 101Ding, H.; Fazelinia, H.; Spruce, L. A.; Weiss, D. A.; Zderic, S. A.; Seeholzer, S. H. Urine Proteomics: Evaluation of Different Sample Preparation Workflows for Quantitative, Reproducible, and Improved Depth of Analysis. J. Proteome Res. 2020, 19 (4), 1857– 1862, DOI: 10.1021/acs.jproteome.9b00772Google Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktlOlsr8%253D&md5=9b16e8ed8bfacc21f7692058946e5d67Urine Proteomics: Evaluation of Different Sample Preparation Workflows for Quantitative, Reproducible, and Improved Depth of AnalysisDing, Hua; Fazelinia, Hossein; Spruce, Lynn A.; Weiss, Dana A.; Zderic, Stephen A.; Seeholzer, Steven H.Journal of Proteome Research (2020), 19 (4), 1857-1862CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)The growing field of urinary proteomics shows promise to expand the no. of biomarkers for the diagnosis and prognosis of a no. of human diseases. With the rapid developments in mass spectrometry methods for proteome quantification, there exists an opportunity for improved sample processing and sepn. workflows to make important contributions to urine proteomic analyses. Here the authors evaluate the performance of four sample prepn. methods: MStern, Pre-Omics in-StageTip (iST), suspension-trapping (S-Trap), and conventional urea In-Soln. trypsin hydrolysis for nondepleted urine samples. Data-dependent acquisition (DDA) mode on a QExactive HF mass spectrometer was used for single-shot label-free data acquisition. The results demonstrate a high degree of reproducibility within each workflow. Pre-Omics iST yields the best digestion efficiency, whereas the S-Trap workflow gives the greatest no. of peptide and protein identifications. Using the S-Trap method and starting with ∼0.5 mL, the authors identify ∼1500 protein groups and ∼17 700 peptides from DDA anal. with a single injection on the mass spectrometer.
- 102Zhang, Z.; Yan, X.; Sun, L.; Zhu, G.; Dovichi, N. J. Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes. Anal. Chem. 2015, 87 (8), 4572– 7, DOI: 10.1021/acs.analchem.5b00789Google Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlsFartbY%253D&md5=ac844ab67b511496c38ce181922ddfc6Detachable Strong Cation Exchange Monolith, Integrated with Capillary Zone Electrophoresis and Coupled with pH Gradient Elution, Produces Improved Sensitivity and Numbers of Peptide Identifications during Bottom-up Analysis of Complex ProteomesZhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J.Analytical Chemistry (Washington, DC, United States) (2015), 87 (8), 4572-4577CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extn. with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic anal. Tryptic digests were prepd. in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concn. but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer vols. while maintaining reasonable peak efficiency and resoln. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by anal. using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h anal., which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. The authors attribute the improved nos. of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. The authors also performed a comparative anal. using a nanoACQUITY UltraPerformance LCH system. Similar nos. of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.
- 103Kasuga, K.; Katoh, Y.; Nagase, K.; Igarashi, K. Microproteomics with microfluidic-based cell sorting: Application to 1000 and 100 immune cells. Proteomics 2017, 17 (13–14), 1600420, DOI: 10.1002/pmic.201600420Google ScholarThere is no corresponding record for this reference.
- 104Zhu, Y.; Piehowski, P. D.; Zhao, R.; Chen, J.; Shen, Y.; Moore, R. J.; Shukla, A. K.; Petyuk, V. A.; Campbell-Thompson, M.; Mathews, C. E.; Smith, R. D.; Qian, W.-J.; Kelly, R. T. Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells. Nat. Commun. 2018, 9 (1), 882, DOI: 10.1038/s41467-018-03367-wGoogle Scholar104https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MrmtlyntQ%253D%253D&md5=ed52404cccbcdc4f8b9081ede1d092cbNanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cellsZhu Ying; Zhao Rui; Kelly Ryan T; Piehowski Paul D; Shen Yufeng; Moore Ronald J; Shukla Anil K; Petyuk Vladislav A; Smith Richard D; Qian Wei-Jun; Chen Jing; Campbell-Thompson Martha; Mathews Clayton ENature communications (2018), 9 (1), 882 ISSN:.Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.
- 105Zhu, Y.; Clair, G.; Chrisler, W. B.; Shen, Y.; Zhao, R.; Shukla, A. K.; Moore, R. J.; Misra, R. S.; Pryhuber, G. S.; Smith, R. D.; Ansong, C.; Kelly, R. T. Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS. Angew. Chem., Int. Ed. 2018, 57 (38), 12370– 12374, DOI: 10.1002/anie.201802843Google Scholar105https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFemur7N&md5=1c6276ba46c3866bfaeb163aee051663Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MSZhu, Ying; Clair, Geremy; Chrisler, William B.; Shen, Yufeng; Zhao, Rui; Shukla, Anil K.; Moore, Ronald J.; Misra, Ravi S.; Pryhuber, Gloria S.; Smith, Richard D.; Ansong, Charles; Kelly, Ryan T.Angewandte Chemie, International Edition (2018), 57 (38), 12370-12374CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors report on the quant. proteomic anal. of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An av. of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. The single-cell proteomics platform can be used to differentiate cell types from enzyme-dissocd. human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.
- 106Cong, Y.; Motamedchaboki, K.; Misal, S. A.; Liang, Y.; Guise, A. J.; Truong, T.; Huguet, R.; Plowey, E. D.; Zhu, Y.; Lopez-Ferrer, D.; Kelly, R. T. Ultrasensitive single-cell proteomics workflow identifies > 1000 protein groups per mammalian cell. Chemical Science 2021, 12 (3), 1001– 1006, DOI: 10.1039/D0SC03636FGoogle Scholar106https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlGjsbfL&md5=1e9df75d659423c2272b7b94626be01fUltrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cellCong, Yongzheng; Motamedchaboki, Khatereh; Misal, Santosh A.; Liang, Yiran; Guise, Amanda J.; Truong, Thy; Huguet, Romain; Plowey, Edward D.; Zhu, Ying; Lopez-Ferrer, Daniel; Kelly, Ryan T.Chemical Science (2021), 12 (3), 1001-1006CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)We report on the combination of nanodroplet sample prepn., ultra-low-flow nanoLC, high-field asym. ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS anal. of multiply charged peptides, resulting in an av. of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential anal. of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.
- 107Leipert, J.; Tholey, A. Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants. Lab Chip 2019, 19 (20), 3490– 3498, DOI: 10.1039/C9LC00715FGoogle Scholar107https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs12it73K&md5=7b70afa31187d549c809b6186681bed7Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactantsLeipert, Jan; Tholey, AndreasLab on a Chip (2019), 19 (20), 3490-3498CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)Digital microfluidics (DMF) is an emerging technique for miniaturized and automated droplet manipulation, which has been proposed as a promising tool for proteomic sample prepn. However, proteome anal. of samples prepd. on-chip by DMF has previously been unfeasible, due to incompatibility with down-stream LC-MS instrumentation. To overcome these limitations, we here developed protocols for bottom-up LC-MS based proteomics sample prepn. of as little as 100 mammalian cells on a com. available digital microfluidics device. To this end, we developed effective cell lysis conditions optimized for DMF, as well as detergent-buffer systems compatible with downstream proteolytic digestion on DMF chips and subsequent LC-MS anal. A major step was the introduction of the single-pot, solid-phase-enhanced sample prepn. (SP3) approach on-chip, which allowed the removal of salts and anti-fouling polymeric detergents, thus rendering sample prepn. by DMF compatible with LC-MS-based proteome anal. Application of DMF-SP3 to the proteome anal. of Jurkat T cells led to the identification of up to 2500 proteins from approx. 500 cells, and up to 1200 proteins from approx. 100 cells on an Orbitrap mass spectrometer, emphasizing the high compatibility of DMF-SP3 with low protein input and minute vols. handled by DMF. Taken together, we demonstrate the first sample prepn. workflow for proteomics on a DMF chip device reported so far, allowing the sensitive anal. of limited biol. material.
- 108Li, Z. Y.; Huang, M.; Wang, X. K.; Zhu, Y.; Li, J. S.; Wong, C. C. L.; Fang, Q. Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis. Anal. Chem. 2018, 90 (8), 5430– 5438, DOI: 10.1021/acs.analchem.8b00661Google Scholar108https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltVGltr8%253D&md5=8afe3849fc4378a687d903d651b983b0Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic AnalysisLi, Zi-Yi; Huang, Min; Wang, Xiu-Kun; Zhu, Ying; Li, Jin-Song; Wong, Catherine C. L.; Fang, QunAnalytical Chemistry (Washington, DC, United States) (2018), 90 (8), 5430-5438CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Single cell proteomic anal. provides crucial information on cellular heterogeneity in biol. systems. Herein, the authors describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic anal. in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, the authors' system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with min. sample loss and a high sample injection efficiency (>99%), thus substantially increasing the anal. sensitivity for single cell samples. The authors applied the present system in the proteomic anal. of 100, 50, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, resp. The OAD chip-based system was further applied in single mouse oocyte anal., with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymic digestion efficiency over the traditional in-tube system.
- 109Lombard-Banek, C.; Reddy, S.; Moody, S. A.; Nemes, P. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS). Mol. Cell Proteomics 2016, 15 (8), 2756– 68, DOI: 10.1074/mcp.M115.057760Google Scholar109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1OktbrL&md5=4cad16002d915c8df43a0452e168bd16Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A.; Nemes, PeterMolecular & Cellular Proteomics (2016), 15 (8), 2756-2768CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance anal. sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technol. builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resoln. mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide sepn., ionization, and data-dependent acquisition, we demonstrate an ~ 75-amol (~ 11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quant. proxy for protein abundance. Correlation anal. was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concn. range.
- 110Liang, Y.; Acor, H.; McCown, M. A.; Nwosu, A. J.; Boekweg, H.; Axtell, N. B.; Truong, T.; Cong, Y.; Payne, S. H.; Kelly, R. T. Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome Profiling. Anal. Chem. 2021, 93 (3), 1658– 1666, DOI: 10.1021/acs.analchem.0c04240Google Scholar110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1CrsL%252FF&md5=c3a012b4d9e2b218ba3589a69a9baae5Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome ProfilingLiang, Yiran; Acor, Hayden; McCown, Michaela A.; Nwosu, Andikan J.; Boekweg, Hannah; Axtell, Nathaniel B.; Truong, Thy; Cong, Yongzheng; Payne, Samuel H.; Kelly, Ryan T.Analytical Chemistry (Washington, DC, United States) (2021), 93 (3), 1658-1666CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Recent advances in sample prepn. and anal. have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prep. and analyze low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized sepn. column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic labs. Here, we report a fully automated platform termed autoPOTS (automated prepn. in one pot for trace samples) that uses only com. available instrumentation for sample processing and anal. An unmodified, low-cost com. robotic pipetting platform was utilized for one-pot sample prepn. We used low-vol. 384-well plates and periodically added water or buffer to the microwells to compensate for limited evapn. during sample incubation. Prepd. samples were analyzed directly from the well plate with a com. autosampler that was modified with a 10-port valve for compatibility with 30μm i.d. nanoLC columns. We used autoPOTS to analyze 1-500 HeLa cells and obsd. only a moderate redn. in peptide coverage for 150 cells and a 24% redn. in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clin. feasibility, we identified an av. of 1095 protein groups from ~ 130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many labs.
- 111Zhou, M.; Uwugiaren, N.; Williams, S. M.; Moore, R. J.; Zhao, R.; Goodlett, D.; Dapic, I.; Pasa-Tolic, L.; Zhu, Y. Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform. Anal. Chem. 2020, 92 (10), 7087– 7095, DOI: 10.1021/acs.analchem.0c00467Google Scholar111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosFWnsrg%253D&md5=2472ebd1f191b4f3419913f0d5999fcaSensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing PlatformZhou, Mowei; Uwugiaren, Naomi; Williams, Sarah M.; Moore, Ronald J.; Zhao, Rui; Goodlett, David; Dapic, Irena; Pasa-Tolic, Ljiljana; Zhu, YingAnalytical Chemistry (Washington, DC, United States) (2020), 92 (10), 7087-7095CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Top-down proteomics is a powerful tool for characterizing genetic variations and post-translational modifications at intact protein level. However, one significant tech. gap of top-down proteomics is the inability to analyze a low amt. of biol. samples, which limits its access to isolated rare cells, fine needle aspiration biopsies, and tissue substructures. Herein, we developed an ultrasensitive top-down platform by incorporating a microfluidic sample prepn. system, termed nanoPOTS (nanodroplet processing in one pot for trace samples), into a top-down proteomic workflow. A unique combination of a nonionic detergent dodecyl-β-D-maltopyranoside (DDM) with urea as protein extn. buffer significantly improved both protein extn. efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing nonspecific adsorption of intact proteins on container surfaces, while urea serves as a strong denaturant to disrupt noncovalent complexes and release intact proteins for downstream anal. The nanoPOTS-based top-down platform reproducibly and quant. identified ~ 170 to ~ 620 proteoforms from ~ 70 to ~ 770 HeLa cells contg. ~ 10 to ~ 115 ng of total protein. A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells. We anticipate the nanoPOTS top-down proteomics platform will be broadly applicable in biomedical research, particularly where clin. specimens are not available in amts. amenable to std. workflows.
- 112Williams, S. M.; Liyu, A. V.; Tsai, C.-F.; Moore, R. J.; Orton, D. J.; Chrisler, W. B.; Gaffrey, M. J.; Liu, T.; Smith, R. D.; Kelly, R. T.; Pasa-Tolic, L.; Zhu, Y. Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography-Mass Spectrometry for High-Throughput Single-Cell Proteomics. Anal. Chem. 2020, 92 (15), 10588– 10596, DOI: 10.1021/acs.analchem.0c01551Google Scholar112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlCmtL3O&md5=11a723a01a532b3fd5dbb8f43fb00134Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography-Mass Spectrometry for High-Throughput Single-Cell ProteomicsWilliams, Sarah M.; Liyu, Andrey V.; Tsai, Chia-Feng; Moore, Ronald J.; Orton, Daniel J.; Chrisler, William B.; Gaffrey, Matthew J.; Liu, Tao; Smith, Richard D.; Kelly, Ryan T.; Pasa-Tolic, Ljiljana; Zhu, YingAnalytical Chemistry (Washington, DC, United States) (2020), 92 (15), 10588-10596CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Single-cell proteomics can provide crit. biol. insight into the cellular heterogeneity that is masked by bulk-scale anal. The authors have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample vols., the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, the authors have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. The authors also developed a sample drying, extn., and loading workflow to enable reproducible and reliable sample injection. The sequential anal. of 20 samples contg. 10 ng tryptic peptides demonstrated high reproducibility with correlation coeffs. of >0.995 between any two samples. The nanoPOTS autosampler can provide anal. throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, resp. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an av. of 256 proteins was identified from each cell and the no. was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ~ 77 single cells could be analyzed per day. The authors analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quant. single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.
- 113Dou, M.; Clair, G.; Tsai, C.-F.; Xu, K.; Chrisler, W. B.; Sontag, R. L.; Zhao, R.; Moore, R. J.; Liu, T.; Pasa-Tolic, L.; Smith, R. D.; Shi, T.; Adkins, J. N.; Qian, W.-J.; Kelly, R. T.; Ansong, C.; Zhu, Y. High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform. Anal. Chem. 2019, 91 (20), 13119– 13127, DOI: 10.1021/acs.analchem.9b03349Google Scholar113https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslKisbbJ&md5=800d1bf514934f3fe80ae23fa44729d7High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation PlatformDou, Maowei; Clair, Geremy; Tsai, Chia-Feng; Xu, Kerui; Chrisler, William B.; Sontag, Ryan L.; Zhao, Rui; Moore, Ronald J.; Liu, Tao; Pasa-Tolic, Ljiljana; Smith, Richard D.; Shi, Tujin; Adkins, Joshua N.; Qian, Wei-Jun; Kelly, Ryan T.; Ansong, Charles; Zhu, YingAnalytical Chemistry (Washington, DC, United States) (2019), 91 (20), 13119-13127CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein the authors combined microfluidic nanodroplet technol. with tandem mass tag (TMT) isobaric labeling to significantly improve anal. throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex anal. of single cell-sized protein quantities to a depth of ∼1600 proteins with median CV of 10.9% and correlation coeff. of 0.98. To demonstrate in-depth high throughput single cell anal., the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2300 proteins identified. Principal component anal. grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. The platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.
- 114Zhu, Y.; Li, H.; Bhatti, S.; Zhou, S.; Yang, Y.; Fish, T.; Thannhauser, T. W. Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots. Hortic. Res. 2016, 3, 16026, DOI: 10.1038/hortres.2016.26Google Scholar114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXms1Kjt7Y%253D&md5=6fa4bfcafc78209bfe741988f8c27323Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant rootsZhu, Yingde; Li, Hui; Bhatti, Sarabjit; Zhou, Suping; Yang, Yong; Fish, Tara; Thannhauser, Theodore W.Horticulture Research (2016), 3 (), 16026/1-16026/8CODEN: HROEA8; ISSN:2052-7276. (Nature Publishing Group)Single-cell-type proteomics provides the capability to revealing the genomic and proteomics information at cell-level resoln. However, the methodol. for this type of research has not been well-developed. This paper reports developing a workflow of laser capture microdissection (LCM) followed by gel-liq. chromatog.-tandem mass spectrometry (GeLC-MS/MS)-based proteomics anal. for the identification of proteomes contained in individual cell layers of tomato roots. Thin-sections (∼10-μm thick, 10 sections per root tip) were prepd. for root tips of tomato germinating seedlings. Epidermal and cortical cells (5000-7000 cells per tissue type) were isolated under a LCM microscope. Proteins were isolated and then sepd. by SDS-polyacrylamide gel electrophoresis followed by in-gel-tryptic digestion. The MS and MS/MS spectra generated using nanoLC-MS/MS anal. of the tryptic peptides were searched against ITAG2.4 tomato protein database to identify proteins contained in each single-cell-type sample. Based on the biol. functions, proteins with proven functions in root hair development were identified in epidermal cells but not in the cortical cells. Several of these proteins were found in Al-treated roots only. The results demonstrated that the cell-type-specific proteome is relevant for tissue-specific functions in tomato roots. Increasing the coverage of proteomes and reducing the inevitable cross-contamination from adjacent cell layers, in both vertical and cross directions when cells are isolated from slides prepd. using intact root tips, are the major challenges using the technol. in proteomics anal. of plant roots.
- 115Zhu, Y.; Dou, M.; Piehowski, P. D.; Liang, Y.; Wang, F.; Chu, R. K.; Chrisler, W. B.; Smith, J. N.; Schwarz, K. C.; Shen, Y.; Shukla, A. K.; Moore, R. J.; Smith, R. D.; Qian, W. J.; Kelly, R. T. Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets. Mol. Cell Proteomics 2018, 17 (9), 1864– 1874, DOI: 10.1074/mcp.TIR118.000686Google Scholar115https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitVKqtL3N&md5=24169c061aaf6216ee6f9d2472e45837Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to nanodropletsZhu, Ying; Dou, Maowei; Piehowski, Paul D.; Liang, Yiran; Wang, Fangjun; Chu, Rosalie K.; Chrisler, William B.; Smith, Jordan N.; Schwarz, Kaitlynn C.; Shen, Yufeng; Shukla, Anil K.; Moore, Ronald J.; Smith, Richard D.; Qian, Wei-Jun; Kelly, Ryan T.Molecular & Cellular Proteomics (2018), 17 (9), 1864-1874CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resoln. because of the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample prepn. system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liq. for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20μm in diam. with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extn. efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on av. from 12-μm-thick rat brain cortex tissue sections having diams. of 50, 100, and 200μm, resp. We also analyzed 100-μm-diam. sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ∼1000 proteins, demonstrating the potential utility for high-resoln. spatially resolved mapping of protein expression in tissues.
- 116Zhu, Y.; Podolak, J.; Zhao, R.; Shukla, A. K.; Moore, R. J.; Thomas, G. V.; Kelly, R. T. Proteome Profiling of 1 to 5 Spiked Circulating Tumor Cells Isolated from Whole Blood Using Immunodensity Enrichment, Laser Capture Microdissection, Nanodroplet Sample Processing, and Ultrasensitive nanoLC-MS. Anal. Chem. 2018, 90 (20), 11756– 11759, DOI: 10.1021/acs.analchem.8b03268Google Scholar116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVeiu7rE&md5=b3ee9305cdd0be798ba76800a602166cProteome Profiling of 1 to 5 Spiked Circulating Tumor Cells Isolated from Whole Blood Using Immunodensity Enrichment, Laser Capture Microdissection, Nanodroplet Sample Processing, and Ultrasensitive nanoLC-MSZhu, Ying; Podolak, Jennifer; Zhao, Rui; Shukla, Anil K.; Moore, Ronald J.; Thomas, George V.; Kelly, Ryan T.Analytical Chemistry (Washington, DC, United States) (2018), 90 (20), 11756-11759CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Proteome profiling of circulating tumor cells (CTCs) can provide crucial insight into disease progression and the role of CTCs in tumor metastasis. The authors describe an integrated workflow to measure global protein expression in 1-5 spiked CTCs enriched from whole blood by immunodensity gradient centrifugation. Enriched CTCs were purified and collected by laser capture microdissection, prepd. using a recently developed nanodroplet-based processing platform (nanoPOTS), and finally analyzed by ultrasensitive nanoLC-MS/MS. The workflow was capable of identifying an av. of 164 and 607 protein groups from samples comprising 1 and 5 LNCaP cells, resp., that were isolated from human whole blood. A panel of prostate cancer-specific proteins were identified and quantified, which was used to differentiate between spiked CTCs and white blood cells.
- 117Dagley, L. F.; Infusini, G.; Larsen, R. H.; Sandow, J. J.; Webb, A. I. Universal Solid-Phase Protein Preparation (USP(3)) for Bottom-up and Top-down Proteomics. J. Proteome Res. 2019, 18 (7), 2915– 2924, DOI: 10.1021/acs.jproteome.9b00217Google Scholar117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVCjtr3L&md5=d6bcccd2b95f1cf8821d76e9a72cafe7Universal Solid-Phase Protein Preparation (USP3) for Bottom-up and Top-down ProteomicsDagley, Laura F.; Infusini, Giuseppe; Larsen, Rune H.; Sandow, Jarrod J.; Webb, Andrew I.Journal of Proteome Research (2019), 18 (7), 2915-2924CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Selecting a sample prepn. strategy for mass spectrometry-based proteomics is crit. to the success of quant. workflows. Here we present a universal, solid-phase protein prepn. (USP3) method which is rapid, robust, and scalable, facilitating high-throughput protein sample prepn. for bottom-up and top-down mass spectrometry (MS) anal. This technique builds upon the single-pot solid-phase-enhanced sample prepn. (SP3) where we now demonstrate its scalability (low to high micrograms of protein) and the influence of variables such as bead and enzyme amts. on the efficiency of protein digestion. We also incorporate acid hydrolysis of DNA and RNA during complete proteome extn. resulting in a more reliable method that is simple and easy to implement for routine and high-throughput anal. of proteins. We benchmarked the performance of this technique against filter-aided sample prepn. (FASP) using 30 μg of total HeLa protein lysate. We also show that the USP3 method is compatible with top-down MS where we reproducibly detect over 1800 proteoforms from 50 μg of HeLa protein lysate. The USP3 protocol allows for efficient and reproducible data to be generated in a cost-effective and robust manner with minimal down time between sample collection and anal. by MS.
- 118Marx, V. A dream of single-cell proteomics. Nat. Methods 2019, 16 (9), 809– 812, DOI: 10.1038/s41592-019-0540-6Google Scholar118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFKhtbfK&md5=f7251798ef90eebb49427b134b45fbe2A dream of single-cell proteomicsMarx, VivienNature Methods (2019), 16 (9), 809-812CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)As single-cell proteomics emerges, perhaps labs can avoid the need to infer protein levels from mRNA abundances.
- 119Cheung, T. K.; Lee, C.-Y.; Bayer, F. P.; McCoy, A.; Kuster, B.; Rose, C. M. Defining the carrier proteome limit for single-cell proteomics. Nat. Methods 2021, 18 (1), 76– 83, DOI: 10.1038/s41592-020-01002-5Google Scholar119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFSltL%252FO&md5=a41f7a517cb6d7999b4c279431cb3187Defining the carrier proteome limit for single-cell proteomicsCheung, Tommy K.; Lee, Chien-Yun; Bayer, Florian P.; McCoy, Atticus; Kuster, Bernhard; Rose, Christopher M.Nature Methods (2021), 18 (1), 76-83CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)Single-cell proteomics by mass spectrometry (SCoPE-MS) is a recently introduced method to quantify multiplexed single-cell proteomes. While this technique has generated great excitement, the underlying technologies (isobaric labeling and mass spectrometry (MS)) have tech. limitations with the potential to affect data quality and biol. interpretation. These limitations are particularly relevant when a carrier proteome, a sample added at 25-500x the amt. of a single-cell proteome, is used to enable peptide identifications. Here we perform controlled expts. with increasing carrier proteome amts. and evaluate quant. accuracy, as it relates to mass analyzer dynamic range, multiplexing level and no. of ions sampled. We demonstrate that an increase in carrier proteome level requires a concomitant increase in the no. of ions sampled to maintain quant. accuracy. Lastly, we introduce Single-Cell Proteomics Companion (SCPCompanion), a software tool that enables rapid evaluation of single-cell proteomic data and recommends instrument and data anal. parameters for improved data quality.
- 120Kostas, J. C.; Gregus, M.; Schejbal, J.; Ray, S.; Ivanov, A. R. Simple and Efficient Microsolid-Phase Extraction Tip-Based Sample Preparation Workflow to Enable Sensitive Proteomic Profiling of Limited Samples (200 to 10,000 Cells). J. Proteome Res. 2021, 20 (3), 1676– 1688, DOI: 10.1021/acs.jproteome.0c00890Google Scholar120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXkvVOksr0%253D&md5=360e8c3240aa1ae49048b458618bbc38Simple and Efficient Microsolid-Phase Extraction Tip-Based Sample Preparation Workflow to Enable Sensitive Proteomic Profiling of Limited Samples (200 to 10,000 Cells)Kostas, James C.; Gregus, Michal; Schejbal, Jan; Ray, Somak; Ivanov, Alexander R.Journal of Proteome Research (2021), 20 (3), 1676-1688CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)In-depth LC-MS-based proteomic profiling of limited biol. and clin. samples, such as rare cells or tissue sections from laser capture microdissection or microneedle biopsies, has been problematic due, in large, to the inefficiency of sample prepn. and attendant sample losses. To address this issue, we developed on-microsolid-phase extn. tip (OmSET)-based sample prepn. for limited biol. samples. OmSET is simple, efficient, reproducible, and scalable and is a widely accessible method for processing ~ 200 to 10,000 cells. The developed method benefits from minimal sample processing vols. (1-3μL) and conducting all sample processing steps on-membrane within a single microreactor. We first assessed the feasibility of using micro-SPE tips for nanogram-level amts. of tryptic peptides, minimized the no. of required sample handling steps, and reduced the hands-on time. We then evaluated the capability of OmSET for quant. anal. of low nos. of human monocytes. Reliable and reproducible label-free quantitation results were obtained with excellent correlations between protein abundances and the amts. of starting material (R2 = 0.93) and pairwise correlations between sample processing replicates (R2 = 0.95) along with the identification of approx. 300, 1800, and 2000 protein groups from injected ~ 10, 100, and 500 cell equiv., resulting from processing approx. 200, 2000, and 10,000 cells, resp.
- 121Chen, P.; Chen, D.; Li, S.; Ou, X.; Liu, B.-F. Microfluidics towards single cell resolution protein analysis. TrAC, Trends Anal. Chem. 2019, 117, 2– 12, DOI: 10.1016/j.trac.2019.06.022Google Scholar121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlaqurfM&md5=f2a7648972c0709a74c6a12fe8cf9260Microfluidics towards single cell resolution protein analysisChen, Peng; Chen, Dongjuan; Li, Shunji; Ou, Xiaowen; Liu, Bi-FengTrAC, Trends in Analytical Chemistry (2019), 117 (), 2-12CODEN: TTAEDJ; ISSN:0165-9936. (Elsevier B.V.)A review. Single cell anal. has aroused great interest with its remarkable ability to investigate cell-to-cell heterogeneity in large populations. However, probing protein information at single cells resoln. including quantity, interactions, and dynamics have been great challenges as a result of the small size of cells, the complexity and a large concn. range of the protein and the lack of genome-wide amplification method. Fortunately, microfluidics capable of high throughput, high reproducibility, large parallelization, easy operability and low-cost, has been emerging as a powerful platform for the anal. of single cell proteins. In this review, we focus the recent advances of microfluidics in single cell resoln. protein anal., particularly covering the following aspects: (1) microfluidic electrophoresis (capillary electrophoresis and gel electrophoresis); (2) microfluidic cytometry (microfluidic flow cytometry, droplet based cytometry and image cytometry); (3) microfluidic array (micro wells, micro chambers, valve-based microfluidics and static droplet array microfluidics); (4) microfluidic probe and (5) microfluidics based mass spectrometry.
- 122He, X.; Chen, Q.; Zhang, Y.; Lin, J.-M. Recent advances in microchip-mass spectrometry for biological analysis. TrAC, Trends Anal. Chem. 2014, 53, 84– 97, DOI: 10.1016/j.trac.2013.09.013Google Scholar122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFyls7jP&md5=2fdb045c17d2089776873d14c9400b72Recent advances in microchip-mass spectrometry for biological analysisHe, Xiangwei; Chen, Qiushui; Zhang, Yandong; Lin, Jin-MingTrAC, Trends in Analytical Chemistry (2014), 53 (), 84-97CODEN: TTAEDJ; ISSN:0165-9936. (Elsevier B. V.)A review. Microchip-mass spectrometry (chip-MS) has been emerging as an excellent anal. tool in the anal. of complex biol. samples. The microchip can play an important role, as in cell culture and sample-prepn. steps prior to mass spectral identification, which benefit from its ability to handle small sample quantities with the potential for high-throughput parallel anal. The authors describe recent progress in chip-MS, including approaches that combined microchip devices with electrospray ionization and matrix-assisted laser desorption/ionization. The authors then review the main applications of chip-MS in proteomics and cell anal. in the past three years. The authors also look at the implications for the future of the field.
- 123Lu, Y.; Yang, L.; Wei, W.; Shi, Q. Microchip-based single-cell functional proteomics for biomedical applications. Lab Chip 2017, 17 (7), 1250– 1263, DOI: 10.1039/C7LC00037EGoogle Scholar123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjs1Gku7o%253D&md5=42b7129f22944e9e78c814ea11b97dc2Microchip-based single-cell functional proteomics for biomedical applicationsLu, Yao; Yang, Liu; Wei, Wei; Shi, QihuiLab on a Chip (2017), 17 (7), 1250-1263CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)Cellular heterogeneity has been widely recognized but only recently have single cell tools become available that allow characterizing heterogeneity at the genomic and proteomic levels. We review the technol. advances in microchip-based toolkits for single-cell functional proteomics. Each of these tools has distinct advantages and limitations, and a few have advanced toward being applied to address biol. or clin. problems that traditional population-based methods fail to address. High-throughput single-cell proteomic assays generate high-dimensional data sets that contain new information and thus require developing new anal. frameworks to ext. new biol. In this review article, we highlight a few biol. and clin. applications in which microchip-based single-cell proteomic tools provide unique advantages. The examples include resolving functional heterogeneity and dynamics of immune cells, dissecting cell-cell interaction by creating a well-controlled on-chip microenvironment, capturing high-resoln. snapshots of immune system functions in patients for better immunotherapy and elucidating phosphoprotein signaling networks in cancer cells for guiding effective molecularly targeted therapies.
- 124Amantonico, A.; Urban, P. L.; Fagerer, S. R.; Balabin, R. M.; Zenobi, R. Single-cell MALDI-MS as an analytical tool for studying intrapopulation metabolic heterogeneity of unicellular organisms. Anal. Chem. 2010, 82 (17), 7394– 400, DOI: 10.1021/ac1015326Google Scholar124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVahsbfP&md5=1155d83d70d9616aea91cd4da6696417Single-Cell MALDI-MS as an Analytical Tool for Studying Intrapopulation Metabolic Heterogeneity of Unicellular OrganismsAmantonico, Andrea; Urban, Pawel L.; Fagerer, Stephan R.; Balabin, Roman M.; Zenobi, RenatoAnalytical Chemistry (Washington, DC, United States) (2010), 82 (17), 7394-7400CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Heterogeneity is a characteristic feature of all populations of living organisms. Here the authors make an attempt to validate a single-cell mass spectrometric method for detection of changes in metabolite levels occurring in populations of unicellular organisms. Selected metabolites involved in central metab. (ADP, ATP, GTP, and UDP-glucose) could readily be detected in single cells of Closterium acerosum by neg.-mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The anal. capabilities of this approach were characterized using std. compds. The method was then used to study populations of individual cells with different levels of the chosen metabolites. With principal component anal. and support vector machine algorithms, it was possible to achieve a clear sepn. of individual C. acerosum cells in different metabolic states. This study demonstrates the suitability of mass spectrometric anal. of metabolites in single cells to measure cell-population heterogeneity.
- 125Korenaga, A.; Chen, F.; Li, H.; Uchiyama, K.; Lin, J.-M. Inkjet automated single cells and matrices printing system for matrix-assisted laser desorption/ionization mass spectrometry. Talanta 2017, 162, 474– 478, DOI: 10.1016/j.talanta.2016.10.055Google Scholar125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVahtr7K&md5=42f9e9b0069a730f1716b16c723f0a5eInkjet automated single cells and matrices printing system for matrix-assisted laser desorption/ionization mass spectrometryKorenaga, Akihito; Chen, Fengming; Li, Haifang; Uchiyama, Katsumi; Lin, Jin-MingTalanta (2017), 162 (), 474-478CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)The ability of single or several cells introduction onto substrate simply would be a useful tool for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this study, we aimed to establish a sample introduction method for pattering cells to the substrate by inkjet technol. Inkjet driving, substrate surface and relative humidity were optimized for single or several cells introduction. Single type cell soln. and MALDI matrix soln. were automatically printed onto ITO glass substrate which was hydrophobic modified under humidity controlled condition. Then the substrate was inserted to MALDI-MS and cells sample soln. provided several peaks from phospholipids. The inkjet technique enables us to print single and subcellular on the substrate with the range of a few hundred micrometers. This diam. would be useful for targeting by laser of MALDI-MS. Our technique provides a new platform for MALDI-MS anal. in single or several cells to get a wide information from one sample.
- 126Yang, M.; Chao, T. C.; Nelson, R.; Ros, A. Direct detection of peptides and proteins on a microfluidic platform with MALDI mass spectrometry. Anal. Bioanal. Chem. 2012, 404 (6–7), 1681– 9, DOI: 10.1007/s00216-012-6257-3Google Scholar126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFWjtL%252FN&md5=b60eebc250432acfb80ceb3948ea4d8cDirect detection of peptides and proteins on a microfluidic platform with MALDI mass spectrometryYang, Mian; Chao, Tzu-Chiao; Nelson, Randall; Ros, AlexandraAnalytical and Bioanalytical Chemistry (2012), 404 (6-7), 1681-1689CODEN: ABCNBP; ISSN:1618-2642. (Springer)The ability to detect and quantify proteins of individual cells in high throughput is of enormous biol. and clin. relevance. Most methods currently in use either require the measurement of large cell populations or are limited to the study of few cells at a time. The authors present the combination of a polydimethylsiloxane-based microfluidic device to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) that allows the detection of as few as 300 mols. at the peptide level and ∼106 to 107 mols. at the protein level. Moreover, the authors performed an immunoassay with subsequent MALDI-TOF-MS to capture and detect insulin immobilized on a surface (∼0.05 mm2) in this device with a detection limit of 106 insulin mols. This microfluidic-based approach therefore begins to approach the sample handling and sensitivity requirements for MS-based single-cell anal. of proteins and peptides and holds the potential for easy parallelization of immunoassays and other highly sensitive protein analyses.
- 127Onjiko, R. M.; Plotnick, D. O.; Moody, S. A.; Nemes, P. Metabolic Comparison of Dorsal versus Ventral Cells Directly in the Live 8-cell Frog Embryo by Microprobe Single-cell CE-ESI-MS. Anal. Methods 2017, 9 (34), 4964– 4970, DOI: 10.1039/C7AY00834AGoogle Scholar127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXntlyrtro%253D&md5=20838177a5e0d4f39a81963871116b0aMetabolic comparison of dorsal versus ventral cells directly in the live 8-cell frog embryo by microprobe single-cell CE-ESI-MSOnjiko, Rosemary M.; Plotnick, David O.; Moody, Sally A.; Nemes, PeterAnalytical Methods (2017), 9 (34), 4964-4970CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)Single-cell mass spectrometry (MS) empowers the characterization of metabolomic changes as cells differentiate to different tissues during early embryogenesis. Using whole-cell dissection and capillary electrophoresis electrospray ionization (CE-ESI) MS, we recently uncovered metabolic cell-to-cell differences in the 8- and 16-cell embryo of the South African clawed frog (Xenopus laevis), raising the question whether metabolic cell heterogeneity is also detectable across the dorsal-ventral axis of the 8-cell embryo. Here, we tested this hypothesis directly in the live embryo by quantifying single-cell metab. between the left dorsal-animal (D1L) and left ventral-animal (V1L) cell pairs in the same embryo using microprobe single-cell CE-ESI-MS in the pos. ion mode. After quantifying ∼70 mol. features, including 52 identified metabolites, that were reproducibly detected in both cells among n = 5 different embryos, we employed supervised multivariate data anal. based on partial least squares discriminant anal. (PLSDA) to compare metab. between the cell types. Statistical anal. revealed that asparagine, glycine betaine, and a yet-unidentified mol. were statistically significantly enriched in the D1L cell compared to V1L (p < 0.05 and fold change ≥ 1.5). These results demonstrate that cells derived from the same hemisphere (animal pole) harbor different metabolic activity along the dorsal-ventral axis as early as the 8-cell stage. Apart from providing new evidence of metabolic cell heterogeneity during early embryogenesis, this study demonstrates that microprobe single-cell CE-ESI-MS enables the anal. of multiple single cells in the same live vertebrate embryo.
- 128Liu, J.; Wang, H.; Manicke, N. E.; Lin, J. M.; Cooks, R. G.; Ouyang, Z. Development, characterization, and application of paper spray ionization. Anal. Chem. 2010, 82 (6), 2463– 71, DOI: 10.1021/ac902854gGoogle Scholar128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhvF2kt7Y%253D&md5=eb5400a81caf7a6f0f69de1526a9f04bDevelopment, Characterization, and Application of Paper Spray IonizationLiu, Jiangjiang; Wang, He; Manicke, Nicholas E.; Lin, Jin-Ming; Cooks, R. Graham; Ouyang, ZhengAnalytical Chemistry (Washington, DC, United States) (2010), 82 (6), 2463-2471CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Paper spray is developed as a direct sampling ionization method for mass spectrometric anal. of complex mixts. Ions of analyte are generated by applying a high voltage to a paper triangle wetted with a small vol. (<10 μL) of soln. Samples can be preloaded onto the paper, added with the wetting soln., or transferred from surfaces using the paper as a wipe. It is demonstrated that paper spray is applicable to the anal. of a wide variety of compds., including small org. compds., peptides, and proteins. Procedures are developed for anal. of dried biofluid spots and applied to therapeutic drug monitoring with whole blood samples and to illicit drug detection in raw urine samples. Limits of detection of 50 ng/mL (or 20 pg abs.) are achieved for atenolol in bovine blood. The combination of sample collection from surfaces and paper spray ionization also enables fast chem. screening at high sensitivity, for example 100 pg of heroin distributed on a surface and agrochems. on fruit peels are detectable. Online derivatization with a preloaded reagent is demonstrated for anal. of cholesterol in human serum. The combination of paper spray with miniature mass spectrometers offers a powerful impetus to wide application of mass spectrometry in nonlab. environments.
- 129Wang, H.; Liu, J.; Cooks, R. G.; Ouyang, Z. Paper spray for direct analysis of complex mixtures using mass spectrometry. Angew. Chem., Int. Ed. 2010, 49 (5), 877– 80, DOI: 10.1002/anie.200906314Google Scholar129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVGmt7g%253D&md5=b6a1e2b784ce8e89d8fa077bc808f66fPaper Spray for Direct Analysis of Complex Mixtures Using Mass SpectrometryWang, He; Liu, Jiangjiang; Cooks, R. Graham; Ouyang, ZhengAngewandte Chemie, International Edition (2010), 49 (5), 877-880, S877/1-S877/7CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The present study reports a paper spray (PS) method which has characteristics of both electrospray ionization and the ambient ionization methods and is useful for fast, qual., and quant. anal. of complex mixts. Analyte transport is achieved by wicking in a porous material with a macroscopically sharp point, and a high elec. field is used to perform ionization. Pneumatic assistance is not required to transport the analyte: a voltage is simply applied to the wet paper, which is held in front of a mass spectrometer.
- 130Wang, H.; Ren, Y.; McLuckey, M. N.; Manicke, N. E.; Park, J.; Zheng, L.; Shi, R.; Cooks, R. G.; Ouyang, Z. Direct quantitative analysis of nicotine alkaloids from biofluid samples using paper spray mass spectrometry. Anal. Chem. 2013, 85 (23), 11540– 11544, DOI: 10.1021/ac402798mGoogle Scholar130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslejsrnP&md5=c4ae341d2353902c2e7fde4e67e85180Direct Quantitative Analysis of Nicotine Alkaloids from Biofluid Samples using Paper Spray Mass SpectrometryWang, He; Ren, Yue; McLuckey, Morgan N.; Manicke, Nicholas E.; Park, Jonghyuck; Zheng, Lingxing; Shi, Riyi; Cooks, R. Graham; Ouyang, ZhengAnalytical Chemistry (Washington, DC, United States) (2013), 85 (23), 11540-11544CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The detn. of tobacco derived nicotine alkaloids in biofluid samples is of great importance to testing for tobacco use, tobacco cessation treatment, and studies on exposure to secondhand smoke. Paper spray mass spectrometry (MS) has been adapted for direct, quant. anal. of tobacco alkaloids from biofluid samples, such as blood, urine, and saliva in liq. and dried form. Limits of quantitation as low as several nanograms per mL were obtained for nicotine, cotinine, trans-3'-hydroxycotinine, and anabasine. Direct anal. of fresh blood samples has also been achieved with improved sensitivity using print paper substrates of high d. Quantitation of the cotinine in the blood of a rat was performed with both direct anal. using paper spray and a traditional anal. protocol using liq. chromatog. MS. Comparable results were obtained and the precision of the two methods was similar. The paper spray MS method is rapid and shows potential for significantly improved anal. efficiency in clin. labs. as well as for point-of-care tobacco use assessment.
- 131Mellors, J. S.; Jorabchi, K.; Smith, L. M.; Ramsey, J. M. Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry. Anal. Chem. 2010, 82 (3), 967– 73, DOI: 10.1021/ac902218yGoogle Scholar131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXivFOltw%253D%253D&md5=2cd158393dfb8ef67fb7826cf08574b0Integrated Microfluidic Device for Automated Single Cell Analysis Using Electrophoretic Separation and Electrospray Ionization Mass SpectrometryMellors, J. Scott; Jorabchi, Kaveh; Smith, Lloyd M.; Ramsey, J. MichaelAnalytical Chemistry (Washington, DC, United States) (2010), 82 (3), 967-973CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)A microfabricated fluidic device was developed for the automated real-time anal. of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free soln. electrophoresis channel, where cellular constituents were sepd., and an integrated electrospray emitter for ionization of sepd. components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in elec. field strength. An electroosmotic pump is incorporated at the end of the electrophoretic sepn. channel to direct eluent to the integrated electrospray emitter. The dissocd. heme group and the α and β subunits of Hb from individual erythrocytes were detected as cells continuously flowed through the device. The av. anal. throughput was approx. 12 cells per min, demonstrating the potential of this method for high-throughput single cell anal.
- 132Chen, Q.; Wu, J.; Zhang, Y.; Lin, J. M. Qualitative and quantitative analysis of tumor cell metabolism via stable isotope labeling assisted microfluidic chip electrospray ionization mass spectrometry. Anal. Chem. 2012, 84 (3), 1695– 701, DOI: 10.1021/ac300003kGoogle Scholar132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XlsVCltA%253D%253D&md5=962b1f1c0ad0aff613cba855b2c8f24dQualitative and Quantitative Analysis of Tumor Cell Metabolism via Stable Isotope Labeling Assisted Microfluidic Chip Electrospray Ionization Mass SpectrometryChen, Qiushui; Wu, Jing; Zhang, Yandong; Lin, Jin-MingAnalytical Chemistry (Washington, DC, United States) (2012), 84 (3), 1695-1701CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)In this work, a stable isotope labeling assisted microfluidic chip electrospray ionization mass spectrometry (SIL-chip-ESI-MS) platform for qual. and quant. anal. of cell metab. was developed. Microfluidic cell culture, drug-induced cell apoptosis anal., and cell metab. measurements were performed simultaneously on the specifically designed device. MCF-7 cells were cultivated in vitro and exposed in anticancer agent (genistein and genistein-d2) for cell-based drug assay. A dual-isotopic labeling was presented for effective qual. anal. of multiplex metabolites. Interestingly, three coeluting pairs of isotopomers appeared with an m/z difference of two. Despite complex biol. matrixes, they can be easily recognized and identified by chip-ESI-MS/MS, which significantly facilitates candidate biomarker discovery. The quant. performance of this system was evaluated using genistein as a model drug by stable isotope diln. anal. The linear equation obtained is y = 0.06x - 3.38 × 10-3 (R2 = 0.995) at the dynamic range from 0.5 to 40 μM. The detection limit is 0.2 μM. The method shows an excellent stability of 2.2% relative std. deviation (RSD) and a good repeatability of 5.5% RSD. The authors' results have successfully demonstrated the capability of selective and quant. anal. of cell-based drug absorption and metabolites with high stability, sensitivity, and repeatability on the chip-ESI-MS system. Consequently, the present device shows promise as a high-throughput, low-cost, and online platform for cell metab. studies and drug screening processes.
- 133Shao, X.; Wang, X.; Guan, S.; Lin, H.; Yan, G.; Gao, M.; Deng, C.; Zhang, X. Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling. Anal. Chem. 2018, 90 (23), 14003– 14010, DOI: 10.1021/acs.analchem.8b03692Google Scholar133https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitVart7%252FO&md5=f7f84b752d5638b2765d395b56eeecf6Integrated Proteome Analysis Device for Fast Single-Cell Protein ProfilingShao, Xi; Wang, Xuantang; Guan, Sheng; Lin, Haizhu; Yan, Guoquan; Gao, Mingxia; Deng, Chunhui; Zhang, XiangminAnalytical Chemistry (Washington, DC, United States) (2018), 90 (23), 14003-14010CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)In the authors' previous work, the authors demonstrated an integrated proteome anal. device (iPAD-100) to analyze proteomes from 100 cells. For the first time, a novel integrated device for single-cell anal. (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 μm internal diam. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL vol., which could prevent protein loss and excessive diln. Digestion was accelerated by using elevated temp. with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead vol. union, to minimize protein transfer loss. A homemade 22 μm internal diam. nano-LC packing column with 3 μm internal diam. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to anal. of the single-cell proteome. Therefore, the total time needed for a single-cell anal. was only 1 h. In an anal. of 10 single HeLa cells, a max. of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estd. at ∼1.7-170 zmol. Such a sensitivity of the iPAD-1 was ∼120-fold higher than that of the authors' previously developed iPAD-100 system. Prominent cellular heterogeneity in protein expressive profiling was obsd. Furthermore, the authors roughly estd. the phases of the cell cycle of tested HeLa cells by the amt. of core histone proteins.
- 134Espina, V.; Milia, J.; Wu, G.; Cowherd, S.; Liotta, L. A. Laser capture microdissection. Methods Mol. Biol. 2006, 319, 213– 29, DOI: 10.1007/978-1-59259-993-6_10Google Scholar134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlCjsb3M&md5=5439af1d3ffdc64a82f3aa1924a8768dLaser capture microdissectionEspina, Virginia; Milia, John; Wu, Glendon; Cowherd, Stacy; Liotta, Lance A.Methods in Molecular Biology (Totowa, NJ, United States) (2006), 319 (Cell Imaging Techniques), 213-229, 1 plateCODEN: MMBIED; ISSN:1064-3745. (Humana Press Inc.)Laser capture microdissection (LCM) is a technique for isolating pure cell populations from a heterogeneous tissue section or cytol. prepn. via direct visualization of the cells. This technique is applicable to mol. profiling of diseased and disease-free tissue, permitting correlation of cellular mol. signatures with specific cell populations. DNA, RNA, or protein anal. can be performed with the microdissected tissue by any method with adequate sensitivity. The principal components of LCM technol. are visualization of the cells of interest via microscopy, transfer of laser energy to a thermolabile polymer with formation of a polymer-cell composite, and removal of the cells of interest from the heterogeneous tissue section. LCM is compatible with a variety of tissue types, cellular staining methods, and tissue-preservation protocols that allow microdissection of fresh or archival specimens. LCM platforms are available as a manual system (PixCell; Arcturus Bioscience) or as an automated system (AutoPix).
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- 1Hwang, B.; Lee, J. H.; Bang, D. Single-cell RNA sequencing technologies and bioinformatics pipelines. Exp. Mol. Med. 2018, 50 (8), 96, DOI: 10.1038/s12276-018-0071-81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c7mtlWlsg%253D%253D&md5=834164f38acc90b870baf3662fafa9f0Single-cell RNA sequencing technologies and bioinformatics pipelinesHwang Byungjin; Bang Duhee; Lee Ji Hyun; Lee Ji HyunExperimental & molecular medicine (2018), 50 (8), 96 ISSN:.Rapid progress in the development of next-generation sequencing (NGS) technologies in recent years has provided many valuable insights into complex biological systems, ranging from cancer genomics to diverse microbial communities. NGS-based technologies for genomics, transcriptomics, and epigenomics are now increasingly focused on the characterization of individual cells. These single-cell analyses will allow researchers to uncover new and potentially unexpected biological discoveries relative to traditional profiling methods that assess bulk populations. Single-cell RNA sequencing (scRNA-seq), for example, can reveal complex and rare cell populations, uncover regulatory relationships between genes, and track the trajectories of distinct cell lineages in development. In this review, we will focus on technical challenges in single-cell isolation and library preparation and on computational analysis pipelines available for analyzing scRNA-seq data. Further technical improvements at the level of molecular and cell biology and in available bioinformatics tools will greatly facilitate both the basic science and medical applications of these sequencing technologies.
- 2Shema, E.; Bernstein, B. E.; Buenrostro, J. D. Single-cell and single-molecule epigenomics to uncover genome regulation at unprecedented resolution. Nat. Genet. 2019, 51 (1), 19– 25, DOI: 10.1038/s41588-018-0290-x2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFCitbjN&md5=2128f6b0bffb7d1c2128cb4fe15ef1baSingle-cell and single-molecule epigenomics to uncover genome regulation at unprecedented resolutionShema, Efrat; Bernstein, Bradley E.; Buenrostro, Jason D.Nature Genetics (2019), 51 (1), 19-25CODEN: NGENEC; ISSN:1061-4036. (Nature Research)A review. Recent advances in single-cell and single-mol. epigenomic technologies now enable the study of genome regulation and dynamics at unprecedented resoln. In this Perspective, we highlight some of these transformative technologies and discuss how they have been used to identify new modes of gene regulation. We also contrast these assays with recent advances in single-cell transcriptomics and argue for the essential role of epigenomic technologies in both understanding cellular diversity and discovering gene regulatory mechanisms. In addn., we provide our view on the next generation of biol. tools that we expect will open new avenues for elucidating the fundamental principles of gene regulation. Overall, this Perspective motivates the use of these high-resoln. epigenomic technologies for mapping cell states and understanding regulatory diversity at single-mol. resoln. within single cells.
- 3Stuart, T.; Satija, R. Integrative single-cell analysis. Nat. Rev. Genet. 2019, 20 (5), 257– 272, DOI: 10.1038/s41576-019-0093-73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXmtFKjtbs%253D&md5=5ca4522fb4d7a0fa547bcfd2333ef4fcIntegrative single-cell analysisStuart, Tim; Satija, RahulNature Reviews Genetics (2019), 20 (5), 257-272CODEN: NRGAAM; ISSN:1471-0056. (Nature Research)The recent maturation of single-cell RNA sequencing (scRNA-seq) technologies has coincided with transformative new methods to profile genetic, epigenetic, spatial, proteomic and lineage information in individual cells. This provides unique opportunities, alongside computational challenges, for integrative methods that can jointly learn across multiple types of data. Integrated anal. can discover relationships across cellular modalities, learn a holistic representation of the cell state, and enable the pooling of data sets produced across individuals and technologies. In this Review, we discuss the recent advances in the collection and integration of different data types at single-cell resoln. with a focus on the integration of gene expression data with other types of single-cell measurement.
- 4Labib, M.; Kelley, S. O. Single-cell analysis targeting the proteome. Nature Reviews Chemistry 2020, 4 (3), 143– 158, DOI: 10.1038/s41570-020-0162-7There is no corresponding record for this reference.
- 5Couvillion, S. P.; Zhu, Y.; Nagy, G.; Adkins, J. N.; Ansong, C.; Renslow, R. S.; Piehowski, P. D.; Ibrahim, Y. M.; Kelly, R. T.; Metz, T. O. New mass spectrometry technologies contributing towards comprehensive and high throughput omics analyses of single cells. Analyst 2019, 144 (3), 794– 807, DOI: 10.1039/C8AN01574K5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlOku77P&md5=90c5aeec3a6c3671a87ae118e424723dNew mass spectrometry technologies contributing towards comprehensive and high throughput omics analyses of single cellsCouvillion, Sneha P.; Zhu, Ying; Nagy, Gabe; Adkins, Joshua N.; Ansong, Charles; Renslow, Ryan S.; Piehowski, Paul D.; Ibrahim, Yehia M.; Kelly, Ryan T.; Metz, Thomas O.Analyst (Cambridge, United Kingdom) (2019), 144 (3), 794-807CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)Mass-spectrometry based omics technologies - namely proteomics, metabolomics and lipidomics - have enabled the mol. level systems biol. investigation of organisms in unprecedented detail. There has been increasing interest for gaining a thorough, functional understanding of the biol. consequences assocd. with cellular heterogeneity in a wide variety of research areas such as developmental biol., precision medicine, cancer research and microbiome science. Recent advances in mass spectrometry (MS) instrumentation and sample handling strategies are quickly making comprehensive omics analyses of single cells feasible, but key breakthroughs are still required to push through remaining bottlenecks. In this review, we discuss the challenges faced by single cell MS-based omics analyses and highlight recent technol. advances that collectively can contribute to comprehensive and high throughput omics analyses in single cells. We provide a vision of the potential of integrating pioneering technologies such as Structures for Lossless Ion Manipulations (SLIM) for improved sensitivity and resoln., novel peptide identification tactics and stds. free metabolomics approaches for future applications in single cell anal.
- 6Hughes, C. S.; Moggridge, S.; Müller, T.; Sorensen, P. H.; Morin, G. B.; Krijgsveld, J. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protoc. 2019, 14 (1), 68– 85, DOI: 10.1038/s41596-018-0082-x6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1yltrzK&md5=d80343142c71d0b55bcd35978e5db19dSingle-pot, solid-phase-enhanced sample preparation for proteomics experimentsHughes, Christopher S.; Moggridge, Sophie; Muller, Torsten; Sorensen, Poul H.; Morin, Gregg B.; Krijgsveld, JeroenNature Protocols (2019), 14 (1), 68-85CODEN: NPARDW; ISSN:1750-2799. (Nature Research)A crit. step in proteomics anal. is the optimal extn. and processing of protein material to ensure the highest sensitivity in downstream detection. Achieving this requires a sample-handling technol. that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually lossless processing. Addressing these needs, the single-pot, solid-phase-enhanced sample-prepn. (SP3) technol. is a paramagnetic bead-based approach for rapid, robust, and efficient processing of protein samples for proteomic anal. SP3 uses a hydrophilic interaction mechanism for exchange or removal of components that are commonly used to facilitate cell or tissue lysis, protein solubilization, and enzymic digestion (e.g., detergents, chaotropes, salts, buffers, acids, and solvents) before downstream proteomic anal. The SP3 protocol consists of nonselective protein binding and rinsing steps that are enabled through the use of ethanol-driven solvation capture on the surface of hydrophilic beads, and elution of purified material in aq. conditions. In contrast to alternative approaches, SP3 combines compatibility with a substantial collection of soln. additives with virtually lossless and unbiased recovery of proteins independent of input quantity, all in a simplified single-tube protocol. The SP3 protocol is simple and efficient, and can be easily completed by a std. user in ∼30 min, including reagent prepn. As a result of these properties, SP3 has successfully been used to facilitate examn. of a broad range of sample types spanning simple and complex protein mixts. in large and very small amts., across numerous organisms. This work describes the steps and extensive considerations involved in performing SP3 in bottom-up proteomics, using a simplified protein cleanup scenario for illustration.
- 7Mao, S.; Li, W.; Zhang, Q.; Zhang, W.; Huang, Q.; Lin, J.-M. Cell analysis on chip-mass spectrometry. TrAC, Trends Anal. Chem. 2018, 107, 43– 59, DOI: 10.1016/j.trac.2018.06.0197https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVersrbN&md5=6087aee9c1f10e2dd530f38c26c1591cCell analysis on chip-mass spectrometryMao, Sifeng; Li, Weiwei; Zhang, Qiang; Zhang, Wanling; Huang, Qiushi; Lin, Jin-MingTrAC, Trends in Analytical Chemistry (2018), 107 (), 43-59CODEN: TTAEDJ; ISSN:0165-9936. (Elsevier B.V.)Benefit from the development of microfluidic chip and mass spectrometry, chip-mass spectrometry (Chip-MS) has become an ambient platform for cell anal. Microfluidic chip has been demonstrated as a powerful tool for cell studies, where identical cells or multiple types of cells were co-cultured and constructed to be an organ or tissue. Improvements of interfaces between microchip and mass spectrometry allow rapid detection and anal. of cell metabolites. Moreover, the Chip-MS platform makes monitoring of cell secretion possible. As the environments on microchip are easy to control, various types of cell researches could be carried out, such as cell metab., cell migration, cell signaling, proteomics and single-cell anal. Here, we review the recent fundamental developments of Chip-MS platform for cell anal. In particular, we discuss the improved integration of Chip-MS platform, as well as its application towards single cell anal.
- 8Yang, L.; George, J.; Wang, J. Deep Profiling of Cellular Heterogeneity by Emerging Single-Cell Proteomic Technologies. Proteomics 2020, 20, e1900226 DOI: 10.1002/pmic.2019002268https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1KisrrL&md5=3d1756a968d1fb35a30ef8ba5f2b1b77Deep Profiling of Cellular Heterogeneity by Emerging Single-Cell Proteomic TechnologiesYang, Liwei; George, Justin; Wang, JunProteomics (2020), 20 (13), 1900226CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biol. responses across a population often loses the information of cellular variations. Single-cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well-established methods for single-cell protein anal. based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining-based techniques for single-cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in anal. of single-cell proteins, along with their biol. applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single-cell proteomic technologies in cell biol. and medical research are discussed.
- 9Kulak, N. A.; Pichler, G.; Paron, I.; Nagaraj, N.; Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 2014, 11 (3), 319– 24, DOI: 10.1038/nmeth.28349https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1Smt78%253D&md5=f2feeabae1635d048cf39b9fbb060e8fMinimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cellsKulak, Nils A.; Pichler, Garwin; Paron, Igor; Nagaraj, Nagarjuna; Mann, MatthiasNature Methods (2014), 11 (3), 319-324CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Mass spectrometry (MS)-based proteomics typically employs multistep sample-prepn. workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed vol. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome anal. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy nos. in these cells were highly correlated (R2 = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-no. ests. for 9,667 human proteins and obsd. excellent quant. reproducibility between replicates (R2 = 0.97). The in-StageTip method is straightforward and generally applicable in biol. or clin. applications.
- 10Scheuermann, S.; Schäfer, A.; Langejürgen, J.; Reis, C. A step towards enzyme-free tissue dissociation. Current Directions in Biomedical Engineering 2019, 5 (1), 545, DOI: 10.1515/cdbme-2019-0137There is no corresponding record for this reference.
- 11Vieira Braga, F. A.; Miragaia, R. J. Tissue Handling and Dissociation for Single-Cell RNA-Seq. Methods Mol. Biol. 2019, 1979, 9– 21, DOI: 10.1007/978-1-4939-9240-9_211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M%252FpslOqsw%253D%253D&md5=120cc9c7389f5888a72f97a285fa3a1cTissue Handling and Dissociation for Single-Cell RNA-SeqVieira Braga Felipe A; Miragaia Ricardo JMethods in molecular biology (Clifton, N.J.) (2019), 1979 (), 9-21 ISSN:.The starting material for all single-cell protocols is a cell suspension. The particular functions and spatial distribution of immune cells generally make them easy to isolate them from the tissues where they dwell. Here we describe tissue dissociation protocols that have been used to obtain human immune cells from lymphoid and nonlymphoid tissues to be then used as input to single-cell methods. We highlight the main factors that can influence the final quality of single-cell data, namely the stress signatures that can bias its interpretation.
- 12Herzenberg, L. A.; Parks, D.; Sahaf, B.; Perez, O.; Roederer, M.; Herzenberg, L. A. The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford. Clin. Chem. 2002, 48 (10), 1819– 1827, DOI: 10.1093/clinchem/48.10.181912https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XnsV2gsLo%253D&md5=6e4a3175ecbb3bc699d3ad6b073a6e99The history and future of the fluorescence activated cell sorter and flow cytometry: a view from StanfordHerzenberg, Leonard A.; Parks, David; Sahaf, Bita; Perez, Omar; Roederer, Mario; Herzenberg, Leonore A.Clinical Chemistry (Washington, DC, United States) (2002), 48 (10), 1819-1827CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)A review. The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells. Becton Dickinson Immunocytometry Systems introduced the com. machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Lab. and a Becton Dickinson engineering group under Bernie Shoor. Over the years, we have increased the no. of measured FACS dimensions (parameters) and the speed of sorting to where we now simultaneously measure 12 fluorescent colors plus 2 scatter parameters. In this history, I illustrate the great utility of this state-of-the-art instrument, which allows us to simultaneously stain, analyze, and then sort cells from small samples of human blood cells from AIDS patients, infants, stem cell transplant patients, and others. I also illustrate anal. and sorting of multiple subpopulations of lymphocytes by use of 8-12 colors. In addn., I review single cell sorting used to clone and analyze hybridomas and discuss other applications of FACS developed over the past 30 yr, as well as give our ideas on the future of FACS. These ideas are currently being implemented in new programs using the internet for data storage and anal. as well as developing new fluorochromes, e.g., green fluorescent protein and tandem dyes, with applications in such areas as apoptosis, gene expression, cytokine expression, cell biochem., redox regulation, and AIDS. Finally, I describe new FACS methods for measuring activated kinases and phosphatases and redox active enzymes in individual cells simultaneously with cell surface phenotyping. Thus, key functions can be studied in various subsets of cells without the need for prior sorting.
- 13Tung, J. W.; Heydari, K.; Tirouvanziam, R.; Sahaf, B.; Parks, D. R.; Herzenberg, L. A.; Herzenberg, L. A. Modern flow cytometry: a practical approach. Clin Lab Med. 2007, 27 (3), 453– 68, DOI: 10.1016/j.cll.2007.05.00113https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2svkt1Oisw%253D%253D&md5=576c5ee1b23035b2bbd7a92d98c74086Modern flow cytometry: a practical approachTung James W; Heydari Kartoosh; Tirouvanziam Rabin; Sahaf Bita; Parks David R; Herzenberg Leonard A; Herzenberg Leonore AClinics in laboratory medicine (2007), 27 (3), 453-68, v ISSN:0272-2712.The demonstration that CD T-cell counts can be used to monitor HIV disease progression opened the way to the first clinical application for fluorescence activated cell sorting (FACS) technology. Modern FACS methodologies such multicolor staining and sorting has opened the way to new and constructive therapeutic and clinical applications. This article outlines approaches in which current users can use to improve the quality of their FACS work without undue effort. FACS technology development and the emergence of new software support for this technology are cooperating in this effort.
- 14Gross, A.; Schoendube, J.; Zimmermann, S.; Steeb, M.; Zengerle, R.; Koltay, P. Technologies for Single-Cell Isolation. Int. J. Mol. Sci. 2015, 16 (8), 16897– 16919, DOI: 10.3390/ijms16081689714https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsl2qsrnI&md5=1ddda4b0fe073c4315d6527fd5dc1759Technologies for single-cell isolationGross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, PeterInternational Journal of Molecular Sciences (2015), 16 (8), 16897-16919CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)The handling of single cells is of great importance in applications such as cell line development or single-cell anal., e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell anal. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) resp. Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/diln. (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Addnl., a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstr. to be relevant to the field.
- 15Arntzen, M. Ø.; Thiede, B. ApoptoProteomics, an integrated database for analysis of proteomics data obtained from apoptotic cells. Molecular & cellular proteomics: MCP 2012, 11 (2), M111.010447, DOI: 10.1074/mcp.M111.010447There is no corresponding record for this reference.
- 16Pawula, M.; Hawthorne, G.; Smith, G. T.; Hill, H. M. Best Practice in Biological Sample Collection, Processing, and Storage for LC-MS in Bioanalysis of Drugs. In Handbook of LC-MS Bioanalysis 2013, 139– 164, DOI: 10.1002/9781118671276.ch13There is no corresponding record for this reference.
- 17Hodge, K.; Have, S. T.; Hutton, L.; Lamond, A. I. Cleaning up the masses: exclusion lists to reduce contamination with HPLC-MS/MS. J. Proteomics 2013, 88, 92– 103, DOI: 10.1016/j.jprot.2013.02.02317https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlsFegu74%253D&md5=0f50a4711381a1ff3fa69c5883a79a73Cleaning up the masses: Exclusion lists to reduce contamination with HPLC-MS/MSHodge, Kelly; Have, Sara Ten; Hutton, Luke; Lamond, Angus I.Journal of Proteomics (2013), 88 (), 92-103CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)Mass spectrometry, in the past five years, has increased in speed, accuracy and use. With the ability of the mass spectrometers to identify increasing nos. of proteins the identification of undesirable peptides (those not from the protein sample) has also increased. Most undesirable contaminants originate in the lab. and come from either the user (e.g. keratin from hair and skin), or from reagents (e.g. trypsin), that are required to prep. samples for anal. We found that a significant amt. of MS instrument time was spent sequencing peptides from abundant contaminant proteins. While completely eliminating non-specific protein contamination is not feasible, it is possible to reduce the sequencing of these contaminants. For example, exclusion lists can provide a list of masses that can be used to instruct the mass spectrometer to 'ignore' the undesired contaminant peptides in the list. We empirically generated be-spoke exclusion lists for several model organisms (Homo sapiens, Caenorhabditis elegans, Saccharomyces cerevisiae and Xenopus laevis), utilizing information from over 500 mass spectrometry runs and cumulative anal. of these data. Here we show that by employing these empirically generated lists, it was possible to reduce the time spent analyzing contaminating peptides in a given sample thereby facilitating more efficient data acquisition and anal. Biol. significanceGiven the current efficacy of the Mass Spectrometry instrumentation, the utilization of data from ~ 500 mass spec runs to generate be-spoke exclusion lists and optimize data acquisition is the significance of this manuscript.T. His article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
- 18Kamlage, B.; Maldonado, S. G.; Bethan, B.; Peter, E.; Schmitz, O.; Liebenberg, V.; Schatz, P. Quality Markers Addressing Preanalytical Variations of Blood and Plasma Processing Identified by Broad and Targeted Metabolite Profiling. Clin. Chem. 2014, 60 (2), 399– 412, DOI: 10.1373/clinchem.2013.21197918https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXisVCisro%253D&md5=ab3494bca4c6bc767a7e37872e43b25aQuality markers addressing preanalytical variations of blood and plasma processing identified by broad and targeted metabolite profilingKamlage, Beate; Maldonado, Sandra Gonzalez; Bethan, Bianca; Peter, Erik; Schmitz, Oliver; Liebenberg, Volker; Schatz, PhilippClinical Chemistry (Washington, DC, United States) (2014), 60 (2), 399-412CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Metabolomics is a valuable tool with applications in almost all life science areas. There is an increasing awareness of the essential need for high-quality biospecimens in studies applying omics technologies and biomarker research. Tools to detect effects of both blood and plasma processing are a key for assuring reproducible and credible results. We report on the response of the human plasma metabolome to common preanal. variations in a comprehensive metabolomics anal. to reveal such high-quality markers. Human EDTA blood was subjected to preanal. variations while being processed to plasma: microclotting, prolonged processing times at different temps., hemolysis, and contamination with buffy layer. In a second expt., EDTA plasma was incubated at different temps. for up to 16 h. Samples were subjected to GC-MS and liq. chromatog.-tandem mass spectrometry-based metabolite profiling (MxP Broad Profiling) complemented by targeted methods, i.e., sphingoids (as part of MxP Lipids), MxP Catecholamines, and MxP Eicosanoids. Short-term storage of blood, hemolysis, and short-term storage of noncooled plasma resulted in statistically significant increases of 4% to 19% and decreases of 8% to 12% of the metabolites. Microclotting, contamination of plasma with buffy layer, and short-term storage of cooled plasma were of less impact on the metabolome (0% to 11% of metabolites increased, 0% to 8% decreased). The response of the human plasma metabolome to preanal. variation demands implementation of thorough quality assurance and QC measures to obtain reproducible and credible results from metabolomics studies. Metabolites identified as sensitive to preanalytics can be used to control for sample quality.
- 19Mitchell, B. L.; Yasui, Y.; Li, C. I.; Fitzpatrick, A. L.; Lampe, P. D. Impact of freeze-thaw cycles and storage time on plasma samples used in mass spectrometry based biomarker discovery projects. Cancer Inf. 2005, 1 (1), 98– 104, DOI: 10.1177/11769351050010011019https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlaksL3F&md5=793e69972fadb54c92f536dea692735eImpact of freeze-thaw cycles and storage time on plasma samples used in mass spectrometry based biomarker discovery projectsMitchell, Breeana L.; Yasui, Yutaka; Li, Christopher I.; Fitzpatrick, Annette L.; Lampe, Paul D.Cancer Informatics (2005), 1 (1), 98-104CODEN: CIANCJ; ISSN:1176-9351. (Libertas Academica Ltd.)Mass spectrometry approaches to biomarker discovery in human fluids have received a great deal of attention in recent years. While mass spectrometry instrumentation and anal. approaches have been widely investigated, little attention has been paid to how sample handling can impact the plasma proteome and therefore influence biomarker discovery. The authors have investigated the effects of two main aspects of sample handling on MALDI-TOF data: repeated freeze-thaw cycles and the effects of long-term storage of plasma at -70°C. Repeated freeze-thaw cycles resulted in a trend towards increasing changes in peak intensity, particularly after two thaws. However, a 4-yr difference in long-term storage appears to have minimal effect on protein in plasma as no differences in peak no., mass distribution, or coeff. of variation were found between samples. Therefore, limiting freeze/thaw cycles seems more important to maintaining the integrity of the plasma proteome than degrdn. caused by long-term storage at -70°C.
- 20Winter, D.; Dehghani, A.; Steen, H. Optimization of Cell Lysis and Protein Digestion Protocols for Protein Analysis by LC-MS/MS. In Proteomic Profiling: Methods and Protocols; Posch, A., Ed.; Springer: New York, NY, 2015; pp 259– 273.There is no corresponding record for this reference.
- 21Shehadul Islam, M.; Aryasomayajula, A.; Selvaganapathy, P. R. A Review on Macroscale and Microscale Cell Lysis Methods. Micromachines 2017, 8 (3), 83, DOI: 10.3390/mi8030083There is no corresponding record for this reference.
- 22Kuhn, A. The Bacterial Cell Wall and Membrane—A Treasure Chest for Antibiotic Targets. In Bacterial Cell Walls and Membranes; Kuhn, A., Ed.; Springer International Publishing: Cham, 2019; pp 1– 5.There is no corresponding record for this reference.
- 23Rose, G. G. A current interpretation of the anatomy of the mammalian cell. In Mammalian Cell Membranes; Jamieson, G. A., Robinson, D. M., Eds.; Butterworth-Heinemann, 1976; pp 1– 30.There is no corresponding record for this reference.
- 24Harayama, T.; Riezman, H. Understanding the diversity of membrane lipid composition. Nat. Rev. Mol. Cell Biol. 2018, 19 (5), 281– 296, DOI: 10.1038/nrm.2017.13824https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit12mt7g%253D&md5=210fa39fb5e39c8020dc9460e7afcba2Understanding the diversity of membrane lipid compositionHarayama, Takeshi; Riezman, HowardNature Reviews Molecular Cell Biology (2018), 19 (5), 281-296CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)A review. Cellular membranes are formed from a chem. diverse set of lipids present in various amts. and proportions. A high lipid diversity is universal in eukaryotes and is seen from the scale of a membrane leaflet to that of a whole organism, highlighting its importance and suggesting that membrane lipids fulfil many functions. Indeed, alterations of membrane lipid homeostasis are linked to various diseases. While many of their functions remain unknown, interdisciplinary approaches have begun to reveal novel functions of lipids and their interactions. It is begin to understand why even small changes in lipid structures and in compn. can have profound effects on crucial biol. functions.
- 25Raffy, S.; Teissié, J. Control of Lipid Membrane Stability by Cholesterol Content. Biophys. J. 1999, 76 (4), 2072– 2080, DOI: 10.1016/S0006-3495(99)77363-725https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXit1Ojs7o%253D&md5=578da1fff74fa00ce3ec4dc40dfda429Control of lipid membrane stability by cholesterol contentRaffy, Sophie; Teissie, JustinBiophysical Journal (1999), 76 (4), 2072-2080CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Cholesterol has a concn.-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane p.d. modulation induced in vesicles when an external elec. field is applied. The magnitude of this modulation is under the control of the membrane elec. permeability. When brought to a crit. value by the external field, the membrane p.d. induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content.
- 26Goldberg, S. Mechanical/physical methods of cell disruption and tissue homogenization. Methods Mol. Biol. 2008, 424, 3– 22, DOI: 10.1007/978-1-60327-064-9_126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1c3htFOntg%253D%253D&md5=495e773d762bad1178d1c6c9246d1138Mechanical/physical methods of cell disruption and tissue homogenizationGoldberg StanleyMethods in molecular biology (Clifton, N.J.) (2008), 424 (), 3-22 ISSN:1064-3745.This chapter covers the various methods of mechanical cell disruption and tissue homogenization that are currently commercially available for processing minute samples (<1 ml) to larger production quantities. These mechanical methods of lysing do not introduce chemicals or enzymes to the system. However, the energies needed when using these "harsh" methods can be high and destroy the very proteins being sought. The destruction of cell membranes and walls is effected by subjecting the cells (1) to shearing by liquid flow, (2) to exploding by pressure differences between inside and outside of cell, (3) to collision forces by impact of beads or paddles, or (4) a combination of these forces. Practical suggestions to optimize each method, where to acquire such equipment, and links to reference sources are included.
- 27Feist, P.; Hummon, A. B. Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int. J. Mol. Sci. 2015, 16 (2), 3537– 63, DOI: 10.3390/ijms1602353727https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXks1eqtb8%253D&md5=e180d65470db5df37df2fd601e57266cProteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samplesFeist, Peter; Hummon, Amanda B.International Journal of Molecular Sciences (2015), 16 (2), 3537-3563CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biol. samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amts. of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amts. of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amts. of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extn., contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liq. chromatog. and bottom-up mass spectrometry-compatible techniques. Also, a range of biol. specimens, including mammalian tissues and model cell culture systems, are discussed.
- 28Cañas, B.; Piñeiro, C.; Calvo, E.; López-Ferrer, D.; Gallardo, J. M. Trends in sample preparation for classical and second generation proteomics. J. Chromatogr A 2007, 1153 (1–2), 235– 58, DOI: 10.1016/j.chroma.2007.01.04528https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXls1Wru78%253D&md5=612a470b0a6f5eb865cc4ff69f397a32Trends in sample preparation for classical and second generation proteomicsCanas, Benito; Pineiro, Carmen; Calvo, Enrique; Lopez-Ferrer, Daniel; Gallardo, Jose ManuelJournal of Chromatography A (2007), 1153 (1-2), 235-258CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)A review. Sample prepn. is a fundamental step in the proteomics workflow. However, it is not easy to find compiled information updating this subject. In this paper, the strategies and protocols for protein extn. and identification, following either classical or second generation proteomics methodologies, are reviewed. Procedures for: tissue disruption, cell lysis, sample pre-fractionation, protein sepn. by 2-DE, protein digestion, mass spectrometry anal., multidimensional peptide sepns. and quantification of protein expression level are described.
- 29DeCaprio, J.; Kohl, T. O. Using Dounce Homogenization to Lyse Cells for Immunoprecipitation. Cold Spring Harb Protoc 2019, 2019 (7), pdb.prot098574, DOI: 10.1101/pdb.prot098574There is no corresponding record for this reference.
- 30Senichkin, V. V.; Prokhorova, E. A.; Zhivotovsky, B.; Kopeina, G. S. Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells. Cells 2021, 10 (4), 852, DOI: 10.3390/cells1004085230https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvF2iu7zP&md5=4a4746ba4ff3b11325669826d294bcc5Simple and efficient protocol for subcellular fractionation of normal and apoptotic cellsSenichkin, Viacheslav V.; Prokhorova, Evgeniia A.; Zhivotovsky, Boris; Kopeina, Gelina S.Cells (2021), 10 (4), 852CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)Subcellular fractionation approaches remain an indispensable tool among a large no. of biochem. methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochem. techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the std. protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the sepn. of nuclear and cytoplasmic contents. The described methodol. is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insol. subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.
- 31Arakawa, T.; Hung, L.; McGinley, M. G.; Rohde, M. F.; Narhi, L. O. Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitor. J. Protein Chem. 1992, 11 (2), 171– 6, DOI: 10.1007/BF0102522231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XksVegur4%253D&md5=ca0cae29c978acb520074799b08303e7Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitorArakawa, Tsutomu; Hung, Lynne; McGinley, Michael G.; Rohde, Michael F.; Narhi, Linda O.Journal of Protein Chemistry (1992), 11 (2), 171-6CODEN: JPCHD2; ISSN:0277-8033.The stabilities of trypsin and soybean trypsin inhibitor in SDS were examd. by SDS-PAGE. Both samples contained several bands, all of which migrated to positions corresponding to the appropriate mol. wt. or less, even when the samples were unheated, suggesting that both the trypsin and trypsin inhibitor are susceptible to SDS-induced denaturation. When they were mixed together prior to addn. of SDS-PAGE sample buffer (1% SDS), a new smearing band appeared which corresponded to a mol. wt. of around 46,000, suggesting that these proteins form a stable complex in SDS. This was confirmed by electroblotting and sequence anal., which indicated that this band contains both the trypsin and inhibitor sequences. At a fixed concn. of the inhibitor, increasing concns. of the trypsin resulted in an increase in the intensity of the complex band. When the mixt. was heated for 10 min in 1% SDS, the complex band disappeared in a temp.-dependent manner. The melting temp. detd. under the exptl. conditions used was about 35°. Similar results were obtained with Bowman-Birk trypsin inhibitor, except that the complex with the above inhibitor had a higher melting temp., around 41°, suggesting that the Bowman-Birk inhibitor/trypsin complex is more stable than the soybean inhibitor/trypsin complex.
- 32Botelho, D.; Wall, M. J.; Vieira, D. B.; Fitzsimmons, S.; Liu, F.; Doucette, A. Top-down and bottom-up proteomics of SDS-containing solutions following mass-based separation. J. Proteome Res. 2010, 9 (6), 2863– 70, DOI: 10.1021/pr900949p32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlsVSks7s%253D&md5=1433f3715c9377beba0d96a1506ca505Top-Down and Bottom-Up Proteomics of SDS-Containing Solutions Following Mass-Based SeparationBotelho, Diane; Wall, Mark J.; Vieira, Douglas B.; Fitzsimmons, Shayla; Liu, Fang; Doucette, AlanJournal of Proteome Research (2010), 9 (6), 2863-2870CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)SDS has recognized benefits for protein sample prepn., including solubilization and imparting mol. wt. sepn. (e.g., SDS-PAGE). Here, we compare two proteome workflows which incorporate SDS for protein sepn., namely, SDS-PAGE coupled to LC/MS (GeLC MS), along with a soln. sepn. platform, GELFrEE, for intact proteome prefractionation and identification. Despite the clear importance of SDS in these and other proteome anal. workflows, the affect of SDS on an LC/MS proteome expt. has not been quantified. The authors first examd. the influence of SDS on both a bottom-up as well as a top-down (intact protein) MS workflow. Surprisingly, at levels up to 0.01% SDS in the injected sample, reliable MS characterization is obtained. The authors subsequently explored org. pptn. protocols (chloroform/methanol/water and acetone) as a means of lowering SDS, and present a simple modified acetone pptn. protocol which consistently enables MS proteome characterizations from samples initially contg. 2% SDS. With this effective strategy for SDS redn., the GELFrEE MS workflow for bottom-up proteome anal. was characterized relative to GeLC MS. Remarkable agreement in the no. and type of identified proteins was obtained from these two sepn. platforms, validating the use of SDS in soln.-phase proteome anal.
- 33Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S.; Gebler, J. C. Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Anal. Chem. 2003, 75 (21), 6023– 8, DOI: 10.1021/ac034619633https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnsVOitr4%253D&md5=b6307096b452612489304dbd1f13e63fEnzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteinsYu, Ying-Qing; Gilar, Martin; Lee, Peter J.; Bouvier, Edouard S. P.; Gebler, John C.Analytical Chemistry (2003), 75 (21), 6023-6028CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Improved in-soln. tryptic digestion of proteins in terms of speed and peptide coverage was achieved with the aid of a novel acid-labile anionic surfactant (ALS). Unlike SDS, ALS solubilizes proteins without inhibiting trypsin or other common endopeptidases activity. Trypsin activity was evaluated in the presence of various denaturants; little or no decrease in proteolytic activity was obsd. in 0.1-1% ALS solns. (w/v). Sample prepn. prior to mass spectrometry and liq. chromatog. anal. consists of sample acidification. ALS degrades rapidly at low-pH conditions, which eliminates surfactant-caused interference with anal. Described methodol. combines the advantages of protein solubilization, rapid digestion, high peptide coverages, and easy sample prepn. for mass spectrometry and liq. chromatog. analyses.
- 34Wiśniewski, J. R. Filter-Aided Sample Preparation for Proteome Analysis. Methods Mol. Biol. 2018, 1841, 3– 10, DOI: 10.1007/978-1-4939-8695-8_134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVeqsbzN&md5=ce0d1d579d508bc0d028fc68b0b57befFilter-aided sample preparation for proteome analysisWisniewski, Jacek R.Methods in Molecular Biology (New York, NY, United States) (2018), 1841 (Microbial Proteomics), 3-10CODEN: MMBIED; ISSN:1940-6029. (Springer)A review. Quant. protein extn. and high-yield generation of peptides from biol. samples are the prerequisite for successful bottom-up type proteomic anal. Filter aided sample prepn. (FASP) is a method for processing of SDS-solubilized cells in a proteomic reactor format. In FASP, disposable centrifugal ultrafiltration units allow for detergent depletion, protein digestion, and isolation of peptides released by proteases from undigested material. Consecutive protein digestion with two or three proteases enables generation of peptide fractions with minimal overlap and considerably increases the no. of identifications and protein sequence coverage. FASP is useful for anal. of samples varying in size from a few micrograms to several milligrams of total protein.
- 35Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 2009, 6 (5), 359– 62, DOI: 10.1038/nmeth.132235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXks12ksb0%253D&md5=f34cb14143462984852497dc1f9ee5c2Universal sample preparation method for proteome analysisWisniewski, Jacek R.; Zougman, Alexandre; Nagaraj, Nagarjuna; Mann, MatthiasNature 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.
- 36Cleland, W. W. Dithiothreitol, a new protective reagent for SH groups. Biochemistry 1964, 3, 480– 2, DOI: 10.1021/bi00892a00236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2cXmvFCqsw%253D%253D&md5=3d97cdc9aaf329681ac68f0b55533469Dithiothreitol, a new protective reagent for SH groupsCleland, W. W.Biochemistry (1964), 3 (4), 480-2CODEN: BICHAW; ISSN:0006-2960.Because of its low oxidn.-redn. potential (-0.33 v. at pH 7), dithiothreitol (and its isomer, dithioerythritol) is capable of maintaining monothiols completely in the reduced state and of reducing disulfides quant. Since this compd. is a highly water-soluble solid with little odor and little tendency to be oxidized directly by air, it should prove much superior to the thiols now used as protective reagents for SH groups.
- 37Deutscher, M. P. Maintaining protein stability. Methods Enzymol. 2009, 463, 121– 7, DOI: 10.1016/S0076-6879(09)63010-X37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt12jsA%253D%253D&md5=2ae1e8f4f54c4e27f261e7b089e3e0f4Maintaining protein stabilityDeutscher, Murray P.Methods in Enzymology (2009), 463 (Guide to Protein Purification), 121-127CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)Proteins are fragile mols. that often require great care during purifn. to ensure that they remain intact and fully active. Nowadays, many proteins are also purified in small amts. under denaturing conditions by various gel electrophoretic techniques, such that inactive proteins are obtained. But even here, it is usually advantageous to maintain the protein in an intact form. In the case of enzymes, and other proteins with assayable biol. activities, maintenance of activity is generally of prime importance, both for following the protein during purifn. and for subsequent studies of function. This chapter will focus on the major points to keep in mind with regard to maintaining the stability of a protein during purifn. and storage. Various other chapters describe in detail stabilization procedures for specific biol. systems and specific classes of proteins.
- 38Hughes, 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 (10), 757, DOI: 10.15252/msb.2014562538https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3ksF2rsA%253D%253D&md5=0a3dce5610fc3f4b8097c6de64ffb9fdUltrasensitive proteome analysis using paramagnetic bead technologyHughes Christopher S; Foehr Sophia; Garfield David A; Furlong Eileen E; Steinmetz Lars M; Krijgsveld JeroenMolecular 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.
- 39Bensaddek, D.; Narayan, V.; Nicolas, A.; Murillo, A. B.; Gartner, A.; Kenyon, C. J.; Lamond, A. I. Micro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level. Proteomics 2016, 16 (3), 381– 392, DOI: 10.1002/pmic.20150026439https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjtFCgsw%253D%253D&md5=cd9d5c69316dee6183da674895bf33abMicro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm levelBensaddek, Dalila; Narayan, Vikram; Nicolas, Armel; Brenes Murillo, Alejandro; Gartner, Anton; Kenyon, Cynthia J.; Lamond, Angus I.Proteomics (2016), 16 (3), 381-392CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)Proteomics studies typically analyze proteins at a population level, using exts. prepd. from tens of thousands to millions of cells. The resulting measurements correspond to av. values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the anal. of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin-assocd. factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (), an open access, searchable database resource.
- 40Noble, J. E.; Bailey, M. J. Quantitation of protein. Methods Enzymol. 2009, 463, 73– 95, DOI: 10.1016/S0076-6879(09)63008-140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt12jsg%253D%253D&md5=496ccb8715492d8e6c0b023259716518Quantitation of proteinNoble, James E.; Bailey, Marc J. A.Methods in Enzymology (2009), 463 (Guide to Protein Purification), 73-95CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)A review. The measurement of protein concn. in an aq. sample is an important assay in biochem. research and development labs for applications ranging from enzymic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly det. the concn. of protein, relative to a std., or using an assigned extinction coeff. Methods are described to provide information on how to analyze protein concn. using UV protein spectroscopy measurements, traditional dye-based absorbance measurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The observation that no single assay dominates the market is due to specific limitations of certain methods that investigators need to consider before selecting the most appropriate assay for their sample. Many of the dye-based assays have unique chem. mechanisms that are prone to interference from chems. prevalent in many biol. buffer prepns. A discussion of which assays are prone to interference and the selection of alternative methods is included.
- 41Sapan, C. V.; Lundblad, R. L.; Price, N. C. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 1999, 29 (2), 99– 10841https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXisFWntbk%253D&md5=f36a96d69924bd2c7d6a421b2b6dd2f7Colorimetric protein assay techniquesSapan, Christine V.; Lundblad, Roger L.; Price, Nicholas C.Biotechnology and Applied Biochemistry (1999), 29 (2), 99-108CODEN: BABIEC; ISSN:0885-4513. (Portland Press Ltd.)A review with 145 refs. There has been an increase in the no. of colorimetric assay techniques for the detn. of protein concn. over the past 20 yr. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coeff. of variation/lab.-to-lab. variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most crit. issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a std. for the calibration of the assay; it is crucial that the std. be representative of the sample. If it is not possible to match the std. with the sample from the perspective of protein compn., then it is preferable to use an assay that is not sensitive to the compn. of the protein such as a micro-Kjeldahl technique, quant. amino acid anal. or the biuret assay. In a complex mixt. it might be inappropriate to focus on a general method of protein detn. and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the "gold std." for a given protein, this method needs to be used routinely for calibration.
- 42Goldring, J. P. Protein quantification methods to determine protein concentration prior to electrophoresis. Methods Mol. Biol. 2012, 869, 29– 35, DOI: 10.1007/978-1-61779-821-4_342https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFWrtrbK&md5=c1eb7012ede468cd48aa78300ff03d0aProtein quantification methods to determine protein concentration prior to electrophoresisGoldring, J. P. DeanMethods in Molecular Biology (New York, NY, United States) (2012), 869 (Protein Electrophoresis), 29-35CODEN: MMBIED; ISSN:1064-3745. (Springer)A review. During each step of a protein isolation technique, if enzyme activity is to be detd. and before a protein mixt. is sepd. on a polyacrylamide electrophoresis gel, it is important to det. the concn. of the protein(s) in soln. Measuring protein concn. involves absorbance in the UV range or staining the protein with dyes or copper. This review describes the various protein detn. methods that can be employed to measure protein concn. in soln.
- 43Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193 (1), 265– 75, DOI: 10.1016/S0021-9258(19)52451-643https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG38XhsVyrsw%253D%253D&md5=19d36395fad7dd87661caa3d2b97640dProtein measurement with the Folin phenol reagentLowry, Oliver H.; Rosebrough, Nira J.; Farr, A. Lewis; Randall, Rose J.Journal of Biological Chemistry (1951), 193 (), 265-75CODEN: JBCHA3; ISSN:0021-9258.cf. C.A. 41, 1271h. Proteins were detd. with the Folin phenol reagent after alk. Cu treatment. The method is as sensitive as with Nessler reagent, yet requires no digestion. It is 10-20 times more sensitive than detn. of the ultraviolet absorption at λ = 280 mμ and is more specific. It is several fold more sensitive than the ninhydrin reaction and 100 times more sensitive than the biuret reaction. Two major disadvantages are: the amt. of color varies with different proteins; the color is not strictly proportional to concn. Few substances cause serious interference. Uric acid, guanine, and xanthine react with Folin reagent. The method is useful for following enzyme fractionation, detg. mixed tissue proteins, detn. of very small amts. of protein, or detg. highly dild. protein, or protein in colored solns. or in the presence of N-contg. material.
- 44Stoscheck, C. M. Quantitation of protein. Methods Enzymol. 1990, 182, 50– 68, DOI: 10.1016/0076-6879(90)82008-P44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXitValsLw%253D&md5=78d0376e0fdafecd901d5e0c9139dc14Quantitation of proteinStoscheck, Christa M.Methods in Enzymology (1990), 182 (Guide Protein Purif.), 50-68CODEN: MENZAU; ISSN:0076-6879.A review with 48 refs. Assays that are easy to perform, require simple instrumentation, and are highly sensitive including methods to conc. samples or to eliminate interfering reagents are discussed.
- 45Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150 (1), 76– 85, DOI: 10.1016/0003-2697(85)90442-745https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlsFKksL0%253D&md5=f4f012331cf4dafa50cdc2d345bade38Measurement of protein using bicinchoninic acidSmith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C.Analytical Biochemistry (1985), 150 (1), 76-85CODEN: ANBCA2; ISSN:0003-2697.A method for the colorimetric detn. of proteins is described which uses bicinchoninic acid to monitor the Cu produced during the biuret reaction. The method (micro and macro) was applied to the detn. of 7 proteins. Bicinchoninic acid forms a 2:1 complex with Cu, resulting in a stable, highly colored chromophore with an absorbance max. at 562 nm. The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concns. When compared to the method of O. H. Lowry et al. (1951), the results reported demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, 1-step anal. and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation assocd. with the Lowry technique.
- 46Desjardins, P.; Hansen, J. B.; Allen, M. Microvolume spectrophotometric and fluorometric determination of protein concentration. Curr. Protoc Protein Sci. 2009, 55, 10, DOI: 10.1002/0471140864.ps0310s55There is no corresponding record for this reference.
Chapter 3, Unit 3.
- 47Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248– 54, DOI: 10.1016/0003-2697(76)90527-347https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XksVehtrY%253D&md5=43f388a493becc4193c7a49d9de0e1dbA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingBradford, Marion M.Analytical Biochemistry (1976), 72 (1-2), 248-54CODEN: ANBCA2; ISSN:0003-2697.A protein detn. method that involves the binding of coomassie Brilliant Blue G 250 to protein is described. The binding of the dye to protein causes a shift in the absorption max. of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm that is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in ∼ 2 min with good color stability for 1 hr. There is little or no interference from cations such as Na+ or K+ nor from carbohydrates such as sucrose. A small amt. of color is developed in the presence of strongly alk. buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amts. of detergents such as Na dodecyl sulfate, Triton X 100, and commercial glassware detergents. Interference by small amts. of detergent may be eliminated by the use of proper control.
- 48Compton, S. J.; Jones, C. G. Mechanism of dye response and interference in the Bradford protein assay. Anal. Biochem. 1985, 151 (2), 369– 74, DOI: 10.1016/0003-2697(85)90190-348https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XjtVag&md5=3445fe9d97f3abff93c1fb8254360b5bMechanism of dye response and interference in the Bradford protein assayCompton, Steve J.; Jones, Clive G.Analytical Biochemistry (1985), 151 (2), 369-74CODEN: ANBCA2; ISSN:0003-2697.The mechanism of Coomassie Brilliant Blue G-250 dye binding in the spectrophotometric detn. of proteins by the method of M. M. Bradford (1976) was examd. with various polyamino acids, amino acids, and natural products. Bradford Coomassie Brilliant Blue G-250 protein-binding dye exists in 3 forms: cationic, neutral, and anionic. Although the anion is not freely present at the dye reagent pH, it is this form that complexes with protein. Dye binding requires a macromol. form with certain reactive functional groups. Interactions are chiefly with arginine rather than primary amino groups; the other basic and arom. residues give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Assay interference by bases, detergents, and other compds. are explained in terms of their effects upon the equil. between the 3 dye forms.
- 49Datki, Z.; Olah, Z.; Macsai, L.; Pakaski, M.; Galik, B.; Mihaly, G.; Kalman, J. Application of BisANS fluorescent dye for developing a novel protein assay. PLoS One 2019, 14 (4), e0215863 DOI: 10.1371/journal.pone.021586349https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFGrtbbF&md5=46990b80832ffd217f9de984ea972d8eApplication of BisANS fluorescent dye for developing a novel protein assayDatki, Zsolt; Olah, Zita; Macsai, Lilla; Pakaski, Magdolna; Galik, Bence; Mihaly, Gabor; Kalman, JanosPLoS One (2019), 14 (4), e0215863CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)In many biol.- and chem.-related research fields and expts. the quantification of the peptide and/or protein concn. in samples are essential. Every research environment has unique requirements, e.g. metal ions, incubation times, photostability, pH, protease inhibitors, chelators, detergents, etc. A new protein assay may be adequate in different expts. beyond or instead of the well-known std. protocols (e.g. Qubit, Bradford or bicinchoninic acid) in related conceptions. Based on our previous studies, we developed a novel protein assay applying the 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (BisANS) fluorescent dye. This mol. has several advantageous properties related to protein detection: good soly. in water, high photostability at adequate pH, quick interaction kinetics (within seconds) with proteins and no exclusionary sensitivity to the chelator, detergent and inhibitor ingredients. The protocol described in this work is highly sensitive in a large spectrum to detect protein (100-fold dild. samples) concns. (from 0.28 up to more than 100 μg/mL). The BisANS protein assay is valid and applicable for quantification of the amt. of protein in different biol. and/or chem. samples.
- 50Gauci, V. J.; Wright, E. P.; Coorssen, J. R. Quantitative proteomics: assessing the spectrum of in-gel protein detection methods. J. Chem. Biol. 2011, 4 (1), 3– 29, DOI: 10.1007/s12154-010-0043-550https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3Mnhslyiuw%253D%253D&md5=c19fc0af6a75a451793dfd84314ef29dQuantitative proteomics: assessing the spectrum of in-gel protein detection methodsGauci Victoria J; Wright Elise P; Coorssen Jens RJournal of chemical biology (2011), 4 (1), 3-29 ISSN:.Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.
- 51Sundaram, R. K.; Balasubramaniyan, N.; Sundaram, P. Protein stains and applications. Methods Mol. Biol. 2012, 869, 451– 64, DOI: 10.1007/978-1-61779-821-4_3951https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFWrt7vJ&md5=84724c904e0cef0afdd35b4c82eccf6aProtein stains and applicationsSundaram, Ranjini K.; Balasubramaniyan, Natarajan; Sundaram, PazhaniMethods in Molecular Biology (New York, NY, United States) (2012), 869 (Protein Electrophoresis), 451-464CODEN: MMBIED; ISSN:1064-3745. (Springer)A review. Staining of proteins sepd. on gels provides the basis for detn. of the crit. properties of these biopolymers, such as their mol. wt. and/or charge. Detection of proteins on gels and blots require stains. These stains vary in sensitivity, ease of use, color, stability, versatility, and specificity. This review discusses different stains and applications with details on how to use the advantages and disadvantages of each stain. It also compiles some important points to be considered in imaging and evaluation. Commonly used colorimetric and fluorescent dyes for general protein staining, and posttranslational modification-specific detection methods are also discussed.
- 52Miller, I.; Crawford, J.; Gianazza, E. Protein stains for proteomic applications: which, when, why?. Proteomics 2006, 6 (20), 5385– 408, DOI: 10.1002/pmic.20060032352https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFyms7nE&md5=a02e2a2858005b4a8c978b7c526fd772Protein stains for proteomic applications: Which, when, why?Miller, Ingrid; Crawford, Johanne; Gianazza, ElisabettaProteomics (2006), 6 (20), 5385-5408CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addn. to theor. considerations, examples are provided to illustrate differential staining of specific proteins with different detection methods. This includes a large body of original data on the comparative evaluation of several pre- and post-electrophoresis stains used in parallel on a single specimen, horse serum run in 2-DE (IPG-DALT). A no. of proteins/protein spots are over- or under-revealed with some of the staining procedures.
- 53Patton, W. F. Detection technologies in proteome analysis. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2002, 771 (1–2), 3– 31, DOI: 10.1016/S1570-0232(02)00043-053https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xks1ylsbo%253D&md5=c961db2f7808477fcc146f3cd9eb7171Detection technologies in proteome analysisPatton, Wayne F.Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2002), 771 (1-2), 3-31CODEN: JCBAAI; ISSN:1570-0232. (Elsevier Science B.V.)A review. Common strategies employed for general protein detection include org. dye, silver stain, radiolabeling, reverse stain, fluorescent stain, chemiluminescent stain and mass spectrometry-based approaches. Fluorescence-based protein detection methods have recently surpassed conventional technologies such as colloidal Coomassie blue and silver staining in terms of quant. accuracy, detection sensitivity, and compatibility with modern downstream protein identification and characterization procedures, such as mass spectrometry. Addnl., specific detection methods suitable for revealing protein post-translational modifications have been devised over the years. These include methods for the detection of glycoproteins, phosphoproteins, proteolytic modifications, S-nitrosylation, arginine methylation and ADP-ribosylation. Methods for the detection of a range of reporter enzymes and epitope tags are now available as well, including those for visualizing β-glucuronidase, β-galactosidase, oligohistidine tags and green fluorescent protein. Fluorescence-based and mass spectrometry-based methodologies are just beginning to offer unparalleled new capabilities in the field of proteomics through the performance of multiplexed quant. anal. The primary objective of differential display proteomics is to increase the information content and throughput of proteomics studies through multiplexed anal. Currently, three principal approaches to differential display proteomics are being actively pursued, difference gel electrophoresis (DIGE), multiplexed proteomics (MP) and isotope-coded affinity tagging (ICAT). New multiplexing capabilities should greatly enhance the applicability of the two-dimensional gel electrophoresis technique with respect to addressing fundamental questions related to proteome-wide changes in protein expression and post-translational modification.
- 54Chevalier, F. Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis. Materials 2010, 3 (10), 4784– 4792, DOI: 10.3390/ma310478454https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlKqtLvP&md5=0bc41583c51d4f7558ba0a2c832acdafStandard dyes for total protein staining in gel-based proteomic analysisChevalier, FrancoisMaterials (2010), 3 (), 4784-4792CODEN: MATEG9; ISSN:1996-1944. (Molecular Diversity Preservation International)A review. Staining of two-dimensional gels is a primary concern in proteomic studies using two-dimensional gel electrophoresis with respect to the no. of proteins analyzed, the accuracy of spot quantification and reproducibility. In this review article, the efficiency of the most widely used dyes was investigated. Visible dyes (Coomassie blue and silver nitrate), fluorescent dyes (Sypro Ruby, Deep Purple) and cyanine labeled methods were compared.
- 55Dyballa, N.; Metzger, S. Fast and sensitive coomassie staining in quantitative proteomics. Methods Mol. Biol. 2012, 893, 47– 59, DOI: 10.1007/978-1-61779-885-6_455https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFWrtL7O&md5=5287dbb77e79706f91fc18011815d12bFast and sensitive Coomassie staining in quantitative proteomicsDyballa, Nadine; Metzger, SabineMethods in Molecular Biology (New York, NY, United States) (2012), 893 (Quantitative Methods in Proteomics), 47-59CODEN: MMBIED; ISSN:1064-3745. (Springer)Proteins sepd. by two-dimensional gel electrophoresis can be visualized by in-gel detection using different staining methods. Ideally, the dye should bind non-covalendy to the protein following a linear response curve. Since protein concns. in biol. systems may vary by six or more orders of magnitude, the staining should allow for a detection of very low protein amts. At the same time, satn. effects have to be avoided because they impede normalized quantification. Most proteomics labs. apply Coomassie, silver, or fluorescent stains. Using the colloidal properties of Coomassie dyes, detection limits at the lower nanogram level can meanwhile be achieved. Characteristics like ease of use, low cost, and compatibility with downstream characterization methods such as mass spectrometry, therefore, make colloidal Coomassie staining well suited for the in-gel detection method in quant. proteomics.
- 56Neuhoff, V.; Stamm, R.; Eibl, H. Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis. Electrophoresis 1985, 6 (9), 427– 448, DOI: 10.1002/elps.115006090556https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlslCqtL8%253D&md5=bd40cbdeb05bf36f77b27744133fd7deClear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysisNeuhoff, Volker; Stamm, Reinhard; Eibl, HansjoergElectrophoresis (1985), 6 (9), 427-48CODEN: ELCTDN; ISSN:0173-0835.A systematic anal. of protein staining was carried out in polyacrylamide gels with Coomassie Brilliant Blue (CBB) R-250 and G-250 by using a high resoln. densitometer allowing for quant. measurements during staining and destaining which revealed that none of the published procedures allows quant. measurements. Protein staining with CBB R-250 in MeOH-H2O-HOAc is poor, as is staining with CBB G-250 in trichloroacetic acid or HClO4, the latter 2, however, allowing for a weak background staining. Consequently using the colloidal properties of the CBB dyes, stronger for G-250 than for R-250, it is possible to increase the sensitivity of protein staining to a detection limit of 0.7 ng bovine serum albumin/mm2 gel. In addn., sensitive protein staining on a clear background is possible. Recipes are described for intensified protein staining with CBB G-250 using richloroacetic acid or HClO4 on a clear background. Optimal staining of proteins on a clear background can be performed with phosphoric acid and CBB G-250 in the presence of (NH4)2SO4 since under these conditions the colloidal state of the dye is optimized. Furthermore, conditions are described which allow the stable fixation of the protein-dye complex. Combining the optimized staining conditions with the stable fixation in 20% (NH4)2SO4 allows for stepwise staining for, e.g., detection of weak spots in addn. to intense protein spots. The dependence of different staining procedures on gel thickness, gel concn., and compds. routinely used in polyacrylamide gel electrophoresis is also analyzed. Calibration curves and application of the new procedure to biol. material demonstrate its wide applicability. Convincing arguments for the colloidal properties of the CBB dyes are presented, formulating the rationale for intensified protein staining with CBB dyes in polyacrylamide gels without background staining.
- 57Chevalier, F.; Rofidal, V.; Rossignol, M. Visible and fluorescent staining of two-dimensional gels. Methods Mol. Biol. 2006, 355, 145– 56, DOI: 10.1385/1-59745-227-0:145There is no corresponding record for this reference.
- 58Harris, L. R.; Churchward, M. A.; Butt, R. H.; Coorssen, J. R. Assessing detection methods for gel-based proteomic analyses. J. Proteome Res. 2007, 6 (4), 1418– 25, DOI: 10.1021/pr070024658https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXivF2isLk%253D&md5=b616525d69ec5378af916cfbaea5d1a9Assessing detection methods for gel-based proteomic analysesHarris, Lauren R.; Churchward, Matthew A.; Butt, R. Hussain; Coorssen, Jens R.Journal of Proteome Research (2007), 6 (4), 1418-1425CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Proteomic analyses using two-dimensional gel electrophoresis (2DE) depend heavily upon the quality of protein stains for sensitive detection. Indeed, detection rather than protein resoln. is likely a current limiting factor in 2DE. The recent development of fluorescent protein stains has dramatically improved the sensitivity of in-gel protein detection and has enabled more accurate protein quantification. Here, we have evaluated the overall quality and relative cost of five com. available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo, and the most commonly used stain, Sypro Ruby (SR). All stains were found to be statistically comparable with regard to no. of protein spots detected, but SR was superior with regard to fluorophore stability (e.g., capacity for repeated use of the stain soln.). Notably, colloidal Coomassie Blue was also found to be comparable to SR when detected using an IR fluorescence imaging system rather than std. densitometry. Thus, depending on available equipment and operating budgets, there are at least two high-sensitivity alternatives to achieve the best currently available in-gel protein detection: Sypro Ruby or Coomassie Blue.
- 59Chevallet, M.; Luche, S.; Rabilloud, T. Silver staining of proteins in polyacrylamide gels. Nat. Protoc. 2006, 1 (4), 1852– 8, DOI: 10.1038/nprot.2006.28859https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFagtrnM&md5=f2489e4f3816dff4767bffff65a46bbbSilver staining of proteins in polyacrylamide gelsChevallet, Mireille; Luche, Sylvie; Rabilloud, ThierryNature Protocols (2006), 1 (4), 1852-1858CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Silver staining is used to detect proteins after electrophoretic sepn. on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chems. It is compatible with downstream processing, such as mass spectrometry anal. after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic sepn. Once completed, the stain is stable for several weeks.
- 60Xie, S.; Wong, A. Y. H.; Kwok, R. T. K.; Li, Y.; Su, H.; Lam, J. W. Y.; Chen, S.; Tang, B. Z. Fluorogenic Ag(+) -Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining. Angew. Chem., Int. Ed. 2018, 57 (20), 5750– 5753, DOI: 10.1002/anie.20180165360https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnt1elsb4%253D&md5=e7bb70fb87fad8fa66d32366f91d0d15Fluorogenic Ag+-Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver StainingXie, Sheng; Wong, Alex Y. H.; Kwok, Ryan T. K.; Li, Ying; Su, Huifang; Lam, Jacky W. Y.; Chen, Sijie; Tang, Ben ZhongAngewandte Chemie, International Edition (2018), 57 (20), 5750-5753CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Silver staining, which exploits the special bioaffinity and the chromogenic redn. of silver ions, is an indispensable visualization method in biol. It is a most popular method for in-gel protein detection. However, it is limited by run-to-run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) anal.; limitations that are largely attributed to the tricky chromogenic visualization. Herein, the authors reported a novel water-sol. fluorogenic Ag+ probe, the sensing mechanism of which is based on an aggregation-induced emission (AIE) process driven by tetrazolate-Ag+ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.
- 61Magdeldin, S.; Enany, S.; Yoshida, Y.; Xu, B.; Zhang, Y.; Zureena, Z.; Lokamani, I.; Yaoita, E.; Yamamoto, T. Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis. Clin. Proteomics 2014, 11 (1), 16, DOI: 10.1186/1559-0275-11-1661https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslShsLnE&md5=5da2864e1ffa73f8e98127a71d5bfa87Basics and recent advances of two dimensional-polyacrylamide gel electrophoresisMagdeldin, Sameh; Enany, Shymaa; Yoshida, Yutaka; Xu, Bo; Zhang, Ying; Zureena, Zam; Lokamani, Ilambarthi; Yaoita, Eishin; Yamamoto, TadashiClinical Proteomics (2014), 11 (), 16/1-16/10, 10 pp.CODEN: CPLRCX; ISSN:1542-6416. (BioMed Central Ltd.)Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional-polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunol. tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its utility still exist. This review discusses the principles of 2-DE as well as both recent methodol. advances and new applications.
- 62Buxbaum, E. Fluorescent Staining of Gels. In Protein Gel Detection and Imaging: Methods and Protocols; Kurien, B. T., Scofield, R. H., Eds.; Springer: New York, NY, 2018; pp 87– 94.There is no corresponding record for this reference.
- 63Basile, F.; Hauser, N. Rapid online nonenzymatic protein digestion combining microwave heating acid hydrolysis and electrochemical oxidation. Anal. Chem. 2011, 83 (1), 359– 67, DOI: 10.1021/ac102470563https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFaktL7P&md5=0e1196140925b31c095cc0649eee6c75Rapid Online Nonenzymatic Protein Digestion Combining Microwave Heating Acid Hydrolysis and Electrochemical OxidationBasile, Franco; Hauser, NicolasAnalytical Chemistry (Washington, DC, United States) (2011), 83 (1), 359-367CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The authors report an online nonenzymic method for site-specific digestion of proteins to yield peptides that are well suited for collision-induced dissocn. tandem mass spectrometry. The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochem. oxidn. at tryptophan and tyrosine. The combined microwave/electrochem. digestion is reproducible and produces peptides with an av. sequence length of 10 amino acids. This peptide length is similar to the av. peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel nonenzymic digestion method, when analyzed by electrospray ionization mass spectrometry, produce protonated mols. with mostly +1 and +2 charge states. The combination of these two nonenzymic methods overcomes shortcomings with each individual method in that (i) peptides generated by the microwave-hydrolysis method have an av. amino acid length of 16 amino acids and (ii) the electrochem.-cleavage method is unable to reproducibly digest proteins with mol. masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min of digestion time) on a series of std. peptides and proteins as well as an Escherichia coli protein ext.
- 64Inglis, A. S. Cleavage at aspartic acid. Methods Enzymol. 1983, 91, 324– 32, DOI: 10.1016/S0076-6879(83)91030-364https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXktValu7c%253D&md5=1202015554ac259c026b4f656d6ffd95Cleavage at aspartic acidInglis, A. S.Methods in Enzymology (1983), 91 (Enzyme Struct., Pt. I), 324-32CODEN: MENZAU; ISSN:0076-6879.The specific cleavage of peptide bonds after aspartic acid residues in proteins by hydrolysis in dil., pH-2 solns. of HCl and HCO2H for 2 h at 108° under vacuum is discussed. Results for the digestion of several proteins all supported the finding that the majority of aspartyl bonds are cleaved after only 2 h.
- 65Rodríguez, J. C.; Wong, L.; Jennings, P. A. The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides. Protein Expression Purif. 2003, 28 (2), 224– 231, DOI: 10.1016/S1046-5928(02)00700-365https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3s3ivFaitA%253D%253D&md5=d7b2a2a6a8ed6b72c59472f905efe90dThe solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptidesRodriguez Juan Carlos; Wong Lilly; Jennings Patricia AProtein expression and purification (2003), 28 (2), 224-31 ISSN:1046-5928.Abnormal fragmentation during cyanogen bromide polypeptide cleavage rarely occurs, although parallel side reactions are known to typically accompany normal cleavage. We have observed that cyanogen bromide cleavage of highly hydrophobic fusion proteins utilized for production of recombinant peptides results in almost complete abolishment of the expected reaction products when the reaction is carried out in 70% trifluoroacetic acid. On the basis of mass spectrometric analysis of the reaction products, we have identified a number of fragments whose origin can be attributed to incomplete fragmentation of the fusion protein, and to unspecific degradation affecting the carrier protein. Substituting the solvent in the reaction media with 70% formic acid or with a matrix composed of 6M guanidinium hydrochloride in 0.1M HCl, however, was found to alleviate polypeptide cleavage. We have attributed the poor yields of the CNBr cleavage carried out in 70% TFA to the increased hydrophobicity of our particular fusion proteins, and to the poor solubilizing ability of this reaction medium. We propose the utilization of chaotropic agents in the presence of diluted acids as the preferred cyanogen bromide cleavage medium of fusion proteins in order to maximize cleavage efficiency of hydrophobic sequences and to prevent deleterious degradation and structural modifications of the target peptides.
- 66Bornstein, P.; Balian, G. Cleavage at Asn-Gly bonds with hydroxylamine. Methods Enzymol. 1977, 47, 132– 45, DOI: 10.1016/0076-6879(77)47016-266https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXltFShurk%253D&md5=9b08e2b28b03950a93105a57588c543dCleavage at Asn-Gly bonds with hydroxylamineBornstein, Paul; Balian, GaryMethods in Enzymology (1977), 47 (Enzyme Struct., Part E), 132-45CODEN: MENZAU; ISSN:0076-6879.A review with 44 refs. This hydroxylaminolysis can be adapted as a relatively specific means for nonenzymic cleavage of the title bonds in proteins.
- 67Chen, L. C.; Kinoshita, M.; Noda, M.; Ninomiya, S.; Hiraoka, K. Rapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MS. J. Am. Soc. Mass Spectrom. 2015, 26 (7), 1085– 91, DOI: 10.1007/s13361-015-1111-467https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlvVersLw%253D&md5=7ee08b76b1f984260e32bde56d4e7e5bRapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MSChen, Lee Chuin; Kinoshita, Masato; Noda, Masato; Ninomiya, Satoshi; Hiraoka, KenzoJournal of the American Society for Mass Spectrometry (2015), 26 (7), 1085-1091CODEN: JAMSEF; ISSN:1044-0305. (Springer)Recently, we reported a new ESI ion source that could electrospray the super-heated aq. soln. with liq. temp. much higher than the normal b.p. (J. Am. Soc. Mass Spectrom.25, 1862-1869). The boiling of liq. was prevented by pressurizing the ion source to a pressure greater than atm. pressure. The max. operating pressure in our previous prototype was 11 atm, and the highest achievable temp. was 180°C. In this paper, a more compact prototype that can operate up to 27 atm and 250°C liq. temps. is constructed, and reproducible MS acquisition can be extended to electrospray temps. that have never before been tested. Here, we apply this super-heated ESI source to the rapid online protein digestion MS. The sample soln. is rapidly heated when flowing through a heated ESI capillary, and the digestion products are ionized by ESI in situ when the soln. emerges from the tip of the heated capillary. With weak acid such as formic acid as soln., the thermally accelerated digestion (acid hydrolysis) has the selective cleavage at the aspartate (Asp, D) residue sites. The residence time of liq. within the active heating region is about 20 s. The online operation eliminates the need to transfer the sample from the digestion reactor, and the output of the digestive reaction can be monitored and manipulated by the soln. flow rate and heater temp. in a near real-time basis. [Figure not available: see fulltext.].
- 68Giansanti, P.; Tsiatsiani, L.; Low, T. Y.; Heck, A. J. R. Six alternative proteases for mass spectrometry-based proteomics beyond trypsin. Nat. Protoc. 2016, 11 (5), 993– 1006, DOI: 10.1038/nprot.2016.05768https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmsFaqsLs%253D&md5=e303807cd5bd00ee3dc461b514c8c6abSix alternative proteases for mass spectrometry-based proteomics beyond trypsinGiansanti, Piero; Tsiatsiani, Liana; Low, Teck Yew; Heck, Albert J. R.Nature Protocols (2016), 11 (5), 993-1006CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics expt. Digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in the authors' ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here the authors provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. Data are presented on protease availability and usage including recommendations for reagent prepn. The authors addnl. describe the appropriate MS data anal. methods and the anticipated results in the case of the anal. of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ∼2 d.
- 69Hustoft, H. K.; Malerød, H.; Wilson, S.; Reubsaet, L.; Lundanes, E.; Greibrokk, T. A Critical Review of Trypsin Digestion for LC-MS Based Proteomics. In Integrative Proteomics; Leung, H.-C., Ed.; InTech, 2012; pp 73– 92.There is no corresponding record for this reference.
- 70Raijmakers, R.; Neerincx, P.; Mohammed, S.; Heck, A. J. R. Cleavage specificities of the brother and sister proteases Lys-C and Lys-N. Chem. Commun. 2009, 46 (46), 8827– 8829, DOI: 10.1039/c0cc02523bThere is no corresponding record for this reference.
- 71Gundry, R. L.; White, M. Y.; Murray, C. I.; Kane, L. A.; Fu, Q.; Stanley, B. A.; Van Eyk, J. E. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Current protocols in molecular biology 2009, 25, DOI: 10.1002/0471142727.mb1025s88There is no corresponding record for this reference.
Chapter 10, Unit 10.
- 72Rappsilber, J.; Ishihama, Y.; Mann, M. Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics. Anal. Chem. 2003, 75 (3), 663– 670, DOI: 10.1021/ac026117i72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XpslOns7w%253D&md5=ae52657518dc8771cdf0868f803e3273Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomicsRappsilber, Juri; Ishihama, Yasushi; Mann, MatthiasAnalytical Chemistry (2003), 75 (3), 663-670CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Proteomics is critically dependent on optimal sample prepn. Particularly, the interface between protein digestion and mass spectrometric anal. has a large influence on the overall quality and sensitivity of the anal. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extn., the procedure has been implemented with com. available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and sepn. material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the prodn. of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 μL/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diam., 0.5 mm length) is 2-4 μg of protein digest, which can be increased by using larger diam. or stacked disks. Five femtomole of tryptic BSA digest could be recovered quant. We have found that the Stage system is well-suited as a universal sample prepn. system for proteomics.
- 73Safarik, I.; Safarikova, M. Magnetic techniques for the isolation and purification of proteins and peptides. BioMagnetic Research and Technology 2004, 2 (1), 7, DOI: 10.1186/1477-044X-2-773https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2sbnvFKjsw%253D%253D&md5=86c8f7ce4fd0ef4c033a02c3b21c9d53Magnetic techniques for the isolation and purification of proteins and peptidesSafarik Ivo; Safarikova MirkaBiomagnetic research and technology (2004), 2 (1), 7 ISSN:1477-044X.Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques.
- 74Waas, M.; Pereckas, M.; Jones Lipinski, R. A.; Ashwood, C.; Gundry, R. L. SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic Analyses. J. Proteome Res. 2019, 18 (4), 1644– 1656, DOI: 10.1021/acs.jproteome.8b0091674https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjsFahtb0%253D&md5=a636f6361d78f63262326dfe2b068208SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic AnalysesWaas, Matthew; Pereckas, Michael; Jones Lipinski, Rachel A.; Ashwood, Christopher; Gundry, Rebekah L.Journal of Proteome Research (2019), 18 (4), 1644-1656CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample prepn. steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among labs. To overcome these challenges, the current study systematically evaluated key exptl. parameters regarding the use of carboxylate-modified paramagnetic particles and detd. those that are crit. for max. performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use std. operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concg. the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides and that the approach is compatible with robotic liq. handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will esp. benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.
- 75Kapoor, K. N.; Barry, D. T.; Rees, R. C.; Dodi, I. A.; McArdle, S. E.; Creaser, C. S.; Bonner, P. L. Estimation of peptide concentration by a modified bicinchoninic acid assay. Anal. Biochem. 2009, 393 (1), 138– 140, DOI: 10.1016/j.ab.2009.06.01675https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptVersbk%253D&md5=722c1fb8c21d94f44fe20de0a02bcf75Estimation of peptide concentration by a modified bicinchoninic acid assayKapoor, Krishan N.; Barry, Danielle T.; Rees, Robert C.; Anthony Dodi, I.; McArdle, Stephanie E. B.; Creaser, Colin S.; Bonner, Philip L. R.Analytical Biochemistry (2009), 393 (1), 138-140CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)Although biuret based protein assays are theor. applicable to peptide measurement, there is a high level of interpeptide variation, detd. largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95° for 5 min in the presence of 0.1 M NaOH contg. 1% (w/v) SDS, prior to incubation for 30 min at 37° in BCA std. working reagent. This modification to the std. bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estn. of the peptide concn. within an unknown sample.
- 76Sargent, M.; Sage, A.; Wolff, C.; Mussell, C.; Neville, D.; Lord, G.; Saeed, M.; Lad, R.; Godfrey, R.; Hird, S.; Barwick, V. Instrumentation. In Guide to Achieving Reliable Quantitative LC-MS Measurements; Sargent, M., Ed.; RSC Analytical Methods Committee, 2013; pp 5– 11.There is no corresponding record for this reference.
- 77Shishkova, E.; Hebert, A. S.; Coon, J. J. Now, More Than Ever, Proteomics Needs Better Chromatography. Cell systems 2016, 3 (4), 321– 324, DOI: 10.1016/j.cels.2016.10.00777Now, More Than Ev