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MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting

2017; Elsevier BV; Volume: 17; Issue: 2 Linguagem: Inglês

10.1074/mcp.o117.067082

ISSN

1535-9484

Autores

Mukesh Kumar, Shai R. Joseph, Martina Augsburg, Aliona Bogdanova, David Drechsel, Nadine L. Vastenhouw, Frank Buchholz, Marc Gentzel, Andrej Shevchenko,

Tópico(s)

Molecular Biology Techniques and Applications

Resumo

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance. Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance. Despite well-known technical limitations and numerous application pitfalls, Western blotting (WB) 1The abbreviations used are: WB, Western blot; FA, formic acid; BSA, bovine serum albumin; CAT, catalase; GP, glycogen phosphorylase; ADH, alcohol dehydrogenase; ENO, enolase; TUBA, alpha-tubulin; UBI, ubiquitin. 1The abbreviations used are: WB, Western blot; FA, formic acid; BSA, bovine serum albumin; CAT, catalase; GP, glycogen phosphorylase; ADH, alcohol dehydrogenase; ENO, enolase; TUBA, alpha-tubulin; UBI, ubiquitin. remains one of the most widely used tools in analytical biochemistry (reviewed in (1.Gorr T.A. Vogel J. Western blotting revisited: critical perusal of underappreciated technical issues.Proteomics Clin. Appl. 2015; 9: 396-405Crossref PubMed Scopus (43) Google Scholar, 2.Gassmann M. Grenacher B. Rohde B. Vogel J. Quantifying Western blots: pitfalls of densitometry.Electrophoresis. 2009; 30: 1845-1855Crossref PubMed Scopus (303) Google Scholar, 3.Baker M. 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Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes.Nat. Protoc. 2006; 1: 1029-1043Crossref PubMed Scopus (304) Google Scholar)); relative proteome-wide quantification using chemical or metabolic labeling (e.g. ICAT (16.Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4341) Google Scholar), TMT (17.Thompson A. Schafer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Johnstone R. Mohammed A.K. Hamon C. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 75: 1895-1904Crossref PubMed Scopus (1709) Google Scholar), iTRAQ (18.Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar, 19.Wiese S. Reidegeld K.A. Meyer H.E. Warscheid B. Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research.Proteomics. 2007; 7: 340-350Crossref PubMed Scopus (594) Google Scholar), SILAC (20.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar), Super SILAC (21.Geiger T. Cox J. Ostasiewicz P. Wisniewski J.R. Mann M. Super-SILAC mix for quantitative proteomics of human tumor tissue.Nat. Methods. 2010; 7: 383-385Crossref PubMed Scopus (432) Google Scholar)), as well as label-free proteome quantification (22.Chelius D. Bondarenko P.V. Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry.J. Proteome Res. 2002; 1: 317-323Crossref PubMed Scopus (345) Google Scholar, 23.Bondarenko P.V. Chelius D. Shaler T.A. Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry.Anal. Chem. 2002; 74: 4741-4749Crossref PubMed Scopus (317) Google Scholar, 24.Ishihama Y. Oda Y. Tabata T. Sato T. Nagasu T. Rappsilber J. Mann M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.Mol. Cell. Proteomics. 2005; 4: 1265-1272Abstract Full Text Full Text PDF PubMed Scopus (1635) Google Scholar, 25.Gao B.B. Stuart L. Feener E.P. Label-free quantitative analysis of 1D-PAGE LC/MS/MS proteome: Application on angiotensin II stimulated smooth muscle cells secretome.Mol. Cell. Proteomics. 2008; 7: 2399-2409Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 26.Wisniewski J.R. Hein M.Y. Cox J. Mann M. A "proteomic ruler" for protein copy number and concentration estimation without spike-in standards.Mol. Cell. Proteomics. 2014; 13: 3497-3506Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar) have been developed. As an alternative to WB of SDS-extracted membrane proteins Arnott et al. developed a method of SRM quantification of preselected pairs of ICAT-labeled peptides enriched by affinity chromatography before LC-MS/MS (27.Arnott D. Kishiyama A. Luis E.A. Ludlum S.G. Marsters J.C. Stults J.T. Selective detection of membrane proteins without antibodies - A mass spectrometric version of the Western blot.Mol. Cell. Proteomics. 2002; 1: 148-156Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). However, these and other developments did not replace WB en masse, although it had been suggested that the field would strongly benefit from routine use of a targeted antibody-independent quantification of proteins by mass spectrometry (28.Mann M. Can proteomics retire the western blot?.J. Proteome Res. 2008; 7: 3065Crossref PubMed Scopus (37) Google Scholar). Because of the attomole sensitivity, protein identification confidence, quantification accuracy, analyses throughput (reviewed in (29.Aebersold R. Mann M. Mass-spectrometric exploration of proteome structure and function.Nature. 2016; 537: 347-355Crossref PubMed Scopus (1105) Google Scholar)) and, last but not least, the availability of high-end mass spectrometers proteomics has had a major impact on the entire field of molecular and cell biology. However, it is often perceived as a tool for monitoring global proteome-wide perturbations that is too cumbersome and inflexible for hypothesis-driven studies encompassing a limited selection of proteins that need to be quantified in many biological conditions. High costs and technical hurdles of proteome-wide labeling of tissues or entire model organisms with stable isotopes; cumbersome preparation of clean protein extracts; inconsistent quality of synthetic peptide standards; biased quantification of membrane and modified proteins are common bottlenecks in targeted proteomics applications. Here we report on a method we termed MS Western that provides multiplexed absolute (in moles) antibody-free quantification of dozens of user-selected proteins from unlabeled cell and tissue lysates that combines sample preparation versatility of conventional WB with the specificity, accuracy, and sensitivity of LC-MS/MS. All reagents were of the analytical grade or better quality. LC-MS grade solvents were purchased from Fisher Scientific (Waltham, MA); formic acid (FA) from Merck (Darmstadt, Germany), Complete Ultra Protease Inhibitors from Roche (Mannheim, Germany); Trypsin Gold, mass spectrometry grade, from Promega (Madison, WI); restriction enzymes and buffers from New England BioLabs (Ipswich, MA); benzonase from Novagen (Gibbstown, NJ); other common chemicals and buffers were from Sigma-Aldrich (Munich, Germany). Precast 4 to 20% gradient 1-mm thick polyacrylamide mini-gels were from Anamed Elektrophorese (Rodau, Germany). Protein standards: bovine serum albumin (BSA), glycogen phosphorylase (GP), alcohol dehydrogenase (ADH), enolase (ENO) and ubiquitin (UBI) were purchased as a lyophilized powder from Sigma-Aldrich. Their purity was checked by 1D SDS-PAGE and by amino acid analysis (Functional Genomics Centre Zurich, Switzerland). Ampoules of Pierce BSA standard and of recombinant human histones were purchased from Thermo Fisher Scientific (Waltham, MA) and from New England BioLabs, respectively. Amino acids were from AppliChem (Darmstadt, Germany); isotopically labeled 13C615N4-l-arginine and 13C6-l-lysine were from Silantes (Munich, Germany). To visualize protein lanes gels were stained with Coomassie CBB R250 and gel slabs covering the mass range of ca ± 20 kDa off the Mr of target proteins were excised. Proteins were in-gel digested with trypsin (30.Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat. Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3531) Google Scholar) and recovered tryptic peptides were analyzed by nanoflow LC-MS/MS (31.Vasilj A. Gentzel M. Ueberham E. Gebhardt R. Shevchenko A. Tissue proteomics by one-dimensional gel electrophoresis combined with label-free protein quantification.J. Proteome Res. 2012; 11: 3680-3689Crossref PubMed Scopus (32) Google Scholar); full details on the GeLC-MS/MS procedure are provided in Supplementary Methods. Mass spectra were acquired in data-dependent acquisition (DDA) mode on LTQ Orbitrap Velos or Q Exactive HF mass spectrometers, both from Thermo Fisher Scientific (Bremen, Germany). DDA settings are provided in supplemental Table S5. To match peptides to target proteins, MS/MS spectra were searched by Mascot v.2.2.04 software (Matrix Science, London, UK) against a customized database containing sequences of all target proteins, human keratins and porcine trypsin (in total, 234 protein entries). We applied precursor mass tolerance of 5 ppm; fragment mass tolerance of 0.6 Da and 0.03Da for LTQ Orbitrap Velos and Q Exactive HF instruments, respectively; fixed modification: carbamidomethyl (C); variable modifications: acetyl (protein N terminus), oxidation (M); labels: 13C(6) (K) and 13C(6)15N(4) (R); cleavage specificity: trypsin, with up to 2 missed cleavages allowed. Peptides having the ions score above 20 were accepted (significance threshold p < 0.05) and the quantification was only carried out if both light and heavy forms of the same peptide were identified by MS/MS and retention time of their XIC peaks matched. Xcalibur (Thermo Fisher Scientific) and Progenesis LC-MS v.4.1 (Nonlinear Dynamics, UK) software were used for extracting peptide features from LC-MS/MS data sets. For benchmarking and validation of MS Western we designed four QconCAT chimeric proteins (CP01 to CP04) having MW in the range of 35 to 264 kDa and comprising proteotypic peptides from proteins of different properties (e.g. cytosolic, transmembrane), size (from 8 to 2065 kDa) and organismal origin. Knock-down experiments in HeLa cells were performed in biological triplicates and analyzed by LC-MS/MS in technical duplicates. MS Western benchmarking experiments were performed in technical duplicates for each of 15 samples made by successive dilution of a total protein extract of HeLa cells. Core histones were quantified in zebrafish embryos in three biological replicates and LC-MS/MS run were acquired in technical duplicates. Wherever applicable, we provide standard deviation (±S.D.), coefficient of variance (CV) and robust coefficient of variance (rCV) calculated as 1.4826 times the median absolute deviation (32.Lawless C. Holman S.W. Brownridge P. Lanthaler K. Harman V.M. Watkins R. Hammond D.E. Miller R.L. Sims P.F. Grant C.M. Eyers C.E. Beynon R.J. Hubbard S.J. Direct and absolute quantification of over 1800 yeast proteins via selected reaction monitoring.Mol. Cell. Proteomics. 2016; 15: 1309-1322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 33.Vizcaíno J. Csordas A. del-Toro N. Dianes J. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q. Wang R. Hermjakob H. 2016 update of the PRIDE database and related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2775) Google Scholar). The overnight culture (OD600 = 1.5) of E. coli strain BL21 (DE3) T1 pRARE was pelleted by centrifugation at 5000 rpm for 5 min at 4 °C (JLA 8.1000 centrifuge from Beckman Coulter, Brea CA). The cell pellet was re-suspended in 2× PBS, lysed in the presence of protease inhibitors and the total protein content was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equimolar mixtures containing 25 fmol to 25 pmol of each protein standard (stock concentrations were quantified by amino acid analysis) were spiked into 50 μg of an E. coli protein extract and subjected to 1D SDS-PAGE as above. On electrophoresis gel slices corresponding to the apparent MWs of spiked proteins were excised, codigested with gel bands of CP01 and recovered peptides quantified by LC-MS/MS. Aliquots of a stock solution of recombinant human H4 with the concentration of 1 mg/ml were appropriately diluted and mixed with equal volumes of 2× Laemmli buffer. Three samples containing 0.05, 0.1, and 0.3 μg of histone H4 were subjected to 1D SDS-PAGE as described above. In parallel, the CP02 and an aliquot containing 0.066 μg of Pierce BSA standard were run on a separate gel. The excised bands of histone H4, CP02, and BSA were mixed and codigested with trypsin. After in-gel codigestion and extraction, tryptic peptides were reconstituted in 46 μl of 5% aqueous FA and 5 μl were analyzed by LC-MS/MS. AKT1, CAT, PLK1, and TUBA4A genes were knock-down (KD) in HeLa cells using RNAi (Eupheria Biotech, Dresden, Germany). KD experiments were performed in triplicate; renilla luciferase transfections as a RNAi specificity control were performed on a separate plate. Further details are provided in Supplementary Methods; details on antibodies and RNAi probes are in supplemental Tables S6 and S7, respectively. HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (Gibco™ Life Technologies). 1 × 107 HeLa cells were re-suspended in 100 μl RIPA buffer containing Complete Protease Inhibitor Mixture, EDTA-free (Roche). Equal volume of 2× Laemmli buffer was added and incubated at 80 °C for 15 min with intermittent vortexing. The supernatant was transferred to a new vial and further diluted with RIPA:2x Laemmli buffer (1:1, v/v). Samples obtained at each dilution step were subjected to 1D SDS-PAGE on two different gels. One gel was analyzed by LI-COR Odyssey and another by MS Western using bands of CP03 and BSA as standard and reference proteins, respectively. In each dilution step an equivalent amount of target proteins was digested and injected into LC-MS/MS or imaged by the Odyssey. Protein extracts of ca. 1 × 107 HeLa cells were prepared as described above, however in one series of experiments, the same amount of cells was homogenized in a twice larger volume of the buffer. Aliquots of cell extracts each equivalent to 4% of the total amount of recovered protein material were loaded onto multiple lanes of polyacrylamide mini-gels. On SDS-PAGE, gel slabs whose Mr corresponded to TUBA and CAT proteins were excised; gel bands of CP03 and of the reference protein BSA were mixed with each gel slab and all samples were in parallel digested with trypsin. After the specified periods of time one sample per each digestion experiment was withdrawn, peptides were extracted from the entire in-gel digest and quantified by LC-MS/MS as described above. Each sample was analyzed in technical duplicates. The amount of protein digested at each time point was calculated by averaging the amounts of five independently quantified peptides from TUBA and BSA and of three peptides from CAT. Wild-type (TLAB) zebrafish embryos were dechorionated immediately on fertilization, synchronized and allowed to develop to the desired stage at 28 °C. Ten embryos per developmental stage (except five embryos for 1-cell stage) were manually deyolked and snap frozen in liquid nitrogen. Samples were boiled in the Laemmli buffer at 98 °C for 10 min and subjected to SDS-PAGE. A single gel slab containing all histones was excised from each sample lane and histones were quantified by MS Western using bands of CP02 and BSA as standard and reference proteins, respectively. 10% of the total amount of recovered tryptic peptides were injected into LC-MS/MS. Zebrafish embryos were collected at the specified developmental stages (n = 5 for H3 and H2B; n = 10 for H2A and H4). Embryos were processed as described above and total protein extracts were subjected to SDS-PAGE. Proteins were blotted onto a nitrocellulose membrane (GE Life Sciences). Primary antibodies (supplemental Table S8) were incubated at room temperature for 1 h or at 4 °C overnight; secondary antibodies (supplemental Table S8) were incubated at room temperature for 45 min. Proteins were quantified by LI-COR Odyssey using tubulin as a loading control. Standards of recombinant human histones were used for making calibration plots for the quantification of corresponding zebrafish homologs. Effectively, MS Western merges the three established analytical approaches: GeLC-MS/MS (30.Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat. Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3531) Google Scholar, 31.Vasilj A. Gentzel M. Ueberham E. Gebhardt R. Shevchenko A. Tissue proteomics by one-dimensional gel electrophoresis combined with label-free protein quantification.J. Proteome Res. 2012; 11: 3680-3689Crossref PubMed Scopus (32) Google Scholar, 34.Cutillas P.R. Biber J. Marks J. Jacob R. Stieger B. Cramer R. Waterfield M. Burlingame A.L. Unwin R.J. Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex.Proteomics. 2004; 5: 101-112Crossref Scopus (54) Google Scholar, 35.Piersma S.R. Warmoes M.O. de Wit M. de Reus I. 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Holman S.W. Brownridge P. Lanthaler K. Harman V.M. Watkins R. Hammond D.E. Miller R.L. Sims P.F. Grant C.M. Eyers C.E. Beynon R.J. Hubbard S.J. Direct and absolute quantification of over 1800 yeast proteins via selected reaction monitoring.Mol. Cell. Proteomics. 2016; 15: 1309-1322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We termed this method as MS Western to underscore that it is targeted (rather than global), quantitative, relies on SDS-PAGE of crude protein extracts and in this way, is in line with classical Western. However, because of mass spectrometry readout, it requires no blotting and, most importantly, no antibodies. To quantify a protein by MS Western we first selected a few (typically, three to six) proteotypic peptides (37.Kuster B. Schirle M. Mallick P. Aebersold R. Scoring proteomes with proteotypic peptide probes.Nat. Rev. Mol. Cell Biol. 2005; 6: 577-583Crossref PubMed Scopus (303) Google Scholar, 39.Mallick P. Schirle M. Chen S.S. Flory M.R. Lee H. Martin D. Ranish J. Raught B. Schmitt R. Werner T. Kuster B. Aebersold R. Computational prediction of proteotypic peptides for quantitative proteomics.Nat. Biotechnol. 2007; 25: 125-131Crossref PubMed Scopus (568) Google Scholar) in preliminary GeLC-MS/MS experiments, which also verified the position of bands of target proteins at the electrophoresis gel lanes separating crude protein extracts. However, peptides could be also picked from collections of LC-MS/MS spectra (43.Craig R. Cortens J.P. Beavis R.C. Open source system for analyzing, validating, and storing protein identification data.J. Proteome Res. 2004; 3: 1234-1242Crossref PubMed Scopus (576) Google Scholar, 44.Desiere F. Deutsch E.W. King N.L. Nesvizhskii A.I. Mallick P. Eng J. Chen S. Eddes J. Loevenich S.N. Aebersold R. The PeptideAtlas project.Nucleic Acids Res. 2006; 34: D655-D658Crossref PubMed Scopus (590) Google Scholar) or predicted by software (45.Lawless C. Hubbard S.J. Prediction of missed proteolytic cleavages for the selection of surrogate peptides for quantitative proteomics.Omics. 2012; 16: 449-456Crossref PubMed Scopus (59) Google Scholar, 46.Eyers C.E. Lawless C. Wedge D.C. Lau K.W. Gaskell S.J. Hubbard S.J. CONSeQuence: prediction of reference peptides for absolute quantitative proteomics using consensus machine learning approaches.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (100) Google Scholar). In the same way, we further selected proteotypic peptides from two reference proteins—in this work we used glycogen phosphorylase (GP) and bovine serum albumin (BSA). Peptide sequences from the target and reference proteins were concatenated in-silico in an arbitrary order except that peptides from the same protein were positioned successively. The entire stretch of peptide sequences was flanked at the N- and C-termini with the sequences of twin-strep-tag followed by a 3C protease cleavage site and His-tag, respectively (Fig. 1). These tags protect target peptides from exopeptidase degradation and, only if deemed necessary, could be used to enrich the expressed chimera from a whole cell lysate. Altogether we designed four project-specific chimera proteins (CP)

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