Quantitative Protein Profiling Using Two-dimensional Gel Electrophoresis, Isotope-coded Affinity Tag Labeling, and Mass Spectrometry

• DOI: 10.1074/mcp.m100013-mcp200
• Publication Date: 2003-04-02T21:49:56Z
• Authors: Marcus Smolka; Huilin Zhou; Ruedi Aebersold
• Keywords: Isobaric labeling; Tandem mass tag; Affinity electrophoresis

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, comprehensively identify quantify the proteins contained in biological samples. We describe a strategy for quantitative based on separation labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis their identification quantification mass spectrometry. The method observation isotopically different precisely co-migrate during therefore two or more encoded samples can be separated concurrently same gel. By analyzing changes proteome yeast (Saccharomyces cerevisiae) induced metabolic shift we show this simple accurately quantifies abundance even cases which multiple migrate to coordinates. particularly useful analysis structural characterization differentially processed post-translationally modified forms expected find wide application research. Proteomics attempts study structure, function, control systems processes systematic many properties proteins. These include sequence (identity), abundance, activity, structure expressed cell, as well modifications, interactions, translocations each might experience. There currently no single experiment platform permits all these proteome-wide scale. Therefore, platforms have been developed specifically subset properties.The complex sample determination relative (quantitative profiling) if are being compared central objectives proteomics. For purpose methods routinely used. first combination established techniques (2DE) 1The abbreviations used are: 2DE, (isoelectric focusing/SDS-PAGE) electrophoresis; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; ICAT™, reagents; IEF, isoelectric focusing; LACB, β-lactoglobulin; LCA, α-lactalbumin; MALDI, matrix-assisted laser desorption-ionization; MS, spectrometry; MW, molecular weight; OVAL, chicken ovalbumin; TOF, time-of-flight. spectrometry (MS) (reviewed Ref. 1Aebersold R. Goodlett D. Mass proteomics.Chem. Rev. 2001; 101: 269-295Google Scholar). second recently procedure (ICAT™) reagent labeling, liquid chromatography, tandem (2Gygi S.P. Rist B. Gerber S.A. Frantisek T. Gelb M.H. Aebersold mixtures using tags.Nat. Biotechnol. 1999; 17: 994-999Google Scholar, 3Griffin T.J. Gygi Loboda A. Jilkine Ens W. Standing K.G. proteomic MALDI quadrupole time-of-flight spectrometer.Anal. Chem. 73: 978-986Google Scholar).At present 2DE/MS most commonly Proteins 2DE detected staining identified, one-by-one, spectrometric peptide fragments derived from protein. supported robust automated instruments perform specific steps process, such imaging, spot picking, digestion, spectrometry, data base searching (1Aebersold 4Traini M. Gooley A.A. Ou K. Wilkins M.R. Tonella L. Sanchez J.-C. Hochstrasser D.F. Williams K.L. Toward approach projects.Electrophoresis. 1998; 19: 1941-1949Google 5Houthaeve Gausepohl H. Ashman Nilson Mann Automated preparation digest robot.J. Protein 1997; 16: 343-348Google 6Wilkins Pennington S.R. Dunn M.J. Proteomics: Sequence Function. Bios, Oxford2001: 171-192Google In achieved image patterns generated gel, implicitly assuming intensity indicates amount spot. 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Difference electrophoresis: detecting extracts.Electrophoresis. 18: 2071-2077Google 12Tonge Shaw J. Middleton Rowlinson Rayner S. Young Pognan F. Hawkins Currie I. Davison Validation differential technology.Proteomics. 1: 377-396Google eliminated at least alleviated some challenges related imaging.As gel-based studies depend it suggested use stable-isotope dilution achieve accurate spectrometer, obviating need imaging (13Oda Y. Huang Cross F.R. Cowburn Chait B.T. Accurate quantitation expression site-specific phosphorylation.Proc. Natl. Acad. Sci. U. 12: 6591-6596Google 20Langen, H., Fountoulakis, M., Evers, S., Wipf, B., Berndt, P. (1998) Proceedings 3rd Siena 2-D Electrophoresis Meeting, Siena, Italy, August 31–September 3, 1998 (Hochstrasser, F., Bini, L., Pallini, V., eds) p. 58, ICES, ItalyGoogle experiments were metabolically stable isotopes (15N 13C), distinguishable isotope signatures combined 2DE. spots then digested, resulting peptides extracted analyzed MS. intensities normal heavy particular was calculate respective Although shown very accurate, cells cultured defined culture conditions.In paper post-isolation isotopic ICAT™ also compatible reagent, determined labeled, cysteine-containing peptides. Both single-step analysis. This reliably identifies containing polypeptides isoform form migrates positions. As chemical proteins, not subsequently 2DE.EXPERIMENTAL PROCEDURESLabeling Standard Proteins—The standard albumin (BSA), ovalbumin (OVAL), β-lactoglobulin (LACB), α-lactalbumin (LCA), superoxide dismutase (Sigma). Labeling performed described previously (14Smolka M.B. Zhou Purkayastha Optimization analysis.Anal. 297: 25-31Google slight modifications. 1 mg dissolved ml Solution (0.05% (w/v) SDS, 6 m urea, 5 mm EDTA, 50 Tris buffer, pH 8.3). Specified amounts solution mixed together (as case under "Results"), aliquots 100 μg total mixture (100 μl) labeled. Disulfide bonds reduced tributyl phosphine (Sigma) 10 min 60 (Applied Biosystems, Framingham, MA) added. (105 nmol) represented ∼1 concentration 3 molar sulfhydryls average 30 kDa 8 cysteine residues per reaction incubated 2 h room temperature Eppendorf tube dark. reactions stopped adding DTT final mm. light reagent-labeled prior separation. Incomplete carried out conditions above except diluted 10-fold, urea omitted.Preparation Yeast Extracts—Yeast cerevisiae strain BWG1-7A) grown until mid-log phase yeast/peptone/dextrose medium either 2% glucose galactose carbon source harvested centrifugation. Glucose- galactose-grown lysed separately lysis buffer 1% Tris, 8.0, phenylmethylsulfonyl fluoride after lyticase treatment prepare spheroblasts according (15Dunn Wobbe C.R. Ausubel F.M. Brent Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl Current Protocols Molecular Biology. Vol. 1. John Wiley & Sons, Inc., New York1987: 13.1-13.9Google An aliquot extracts determine commercial assay kit (Bio-Rad). To remainder added mm, boiled min. 300 extract precipitated cold ethanol:acetone:acetic acid (50:50:0.1 (v/v/v)) solution, precipitate washed once 70% ethanol water. pellet re-suspended μl A, disulfide addition incubation temperature. (∼3 concentration) solution. After mm.For comparing (glucose- galactose-grown) again above, re-dissolved, both subsequent equal combined, 2DE.Two-dimensional Gel Electrophoresis—Approximately 20 solubilized 340 4% (v/v) carrier ampholytes, 3–10, 70 0.001% bromphenol blue. Except Sigma, Amersham Biosciences, Inc. Samples applied immobilized gradient strips non-linear 3–10 (catalog number 17-1235-01; Inc.). 10-h rehydration, focusing out, °C, 500 V, additional hour 1000 8000 V IPGphor apparatus (Amersham Inc.) maintaining limiting current μA strip. First dimension subjected reduction alkylation second-dimension electrophoresis. Strips soaked Tris-HCl, 6.8, 30% glycerol, solvent 2.5% iodoacetamide instead DTT. Second-dimension (SDS-PAGE) SE 600 connected MultiTemp II refrigerating (both laying strip top 12.5% polyacrylamide sealing agarose, 90 time constant amperage mA dye front reached lower end nitrate protocol adapted 16Blum Beier Gross H.J. Electrophoresis. 1987; 8: 93-99Google Scholar.Protein Identification Quantitation Matrix-assisted Laser Desorption Ionization (MALDI) Quadrupole Time-of-Flight (TOF) MS—Peptides gel-separated following in-gel tryptic digestion protocols (17Schevechenko Wilm Vorm O. sequencing silver-stained gels.Anal. 68: 850-858Google QSTAR™ Pulsar TOF spectrometer Biosystems/MDS Sciex, Toronto, Canada), equipped ion source. Prior plate purified C18 ZipTips (Millipore) 1:1 16% 2,5-dihydrobenzoic acetonitrile:water (25:75 v/v).Identification submitting its known sequences Peptidemass tool (18Wilkins Lindskog Gasteiger Bairoch Appel R.D. Detailed PEPTIDEMASS-a World-Wide Web accessible tool.Electrophoresis. 403-408Google manually predicted masses measured spectrum. extracts, searched against entries Swiss-Prot MS-Fit program (University California, San Francisco; prospector.ucsf.edu/) (19Clauser K.R. Baker P Burlingame A.L. Role measurement (± ppm) strategies employing MS MS/MS database searching.Anal. 71: 2871-2882Google program, already possible consider cysteines calculating observed pI MW identified map theoretical confirm identifications.For quantifying protein, peak height monoisotopic ICAT™-labeled divided peptide. When than one calculated pair highest intensity. shape distributions ICAT™–labeled differed (i.e. asymmetric clusters) suspected presence contaminating peaks did pairs quantification. Instead, next used.RESULTSPrinciple Method—The schematically illustrated Fig. It separate (non-deuterated) (deuterated), 14Smolka 2T. Nadler, Parker, Wagenfeld, Lotti, Purkayastha, Daniels, Stanick, Pillai, N. Marchese, G. Vella (2001) preparing submitted publication. protocol. migrating enzymatically digested matrix, mapping single-stage (21Henzel W.J. Billeci T.M. Stults J.T. Wong S.C. Grimley C. Watanabe Identifying databases.Proc. 1993; 90: 5011-5015Google 22Pappin D.J. Peptide fingerprinting MALDI-TOF spectrometry.Methods Mol. Biol. 64: 165-173Google collision-induced dissociation selected (23Yates III, J.R. Eng J.K. McCormack Schieltz Method correlate spectra amino database.Anal. 1995; 67: 1426-1436Google protein(s) specific, tagged peptide.Migration Reagent-labeled 2DE—To whether how affected migration LACB LCA profile obtained unlabeled but otherwise identical conditions. 2, B profiles samples, respectively. apparent decreased electrophoretic mobility decrease consistent 442 Da molecule. contains eight cysteines, increased 14186 17722 Da, 14 ∼18 kDa. five 18281 20491 18 ∼21 contrast, consequence labeling. acidic unaffected fact molecule uncharged and, reacting sulfhydryl groups values, does alter charge state basic change occur negative values pK will neutralized reaction.Fig. 2Effect migration. (A), completely (B), partially (C) (LACB) (LCA) shown.View Large Image Figure ViewerDownload (PPT)We demonstrated incomplete results "fuzzy" banding SDS-PAGE reported optimized complete 2C shows non-optimized evident easily recognized characteristic ladder success method.Co-migration Labeled Isotopically Normal Heavy Reagents—To migrated coordinates reagent. d0:d8 (light ICAT™: ICAT™) 11:1, spot, staining, into four quadrants, quadrant spectrometrically. 3A quadrants 3B detail ratios 11.00. averaging 11.72. deviation mean value 11.30, corresponding 3.6% difference.Fig. 3Co-migration Aliquots 11:1 dissected trypsinized, orientation numbering sections indicated B.View chose experiment, prevalence would exaggerate effects At 64 deuterium atoms. Moreover, (4.8) (14.1 kDa) position area high resolution indicate 2DE.Accuracy Quantification—To evaluate accuracy prepared quantities. composition Table One other trypsin, quantified spectrometry.TABLE IComposition 5ProteinMixture (d0-labeled)Mixture (d8-labeled)Expected ratioObserved ratioErrorμgd0:d8%Superoxide (SOD)34172.00:1.002.02:1.001LCA44411.00:1.0011.72:1.007.92LACB9201.00:2.221.00:2.124.4OVAL7351.00:5.001.00:5.510BSA6241.00:4.001.00:3.5712 Open table tab concise 2D electropherogram absence vertical "streaking" had (Fig. 4A). (with pI) BSA (data shown) unrelated identity confirmed fingerprinting. spectra. 4B spectrum expansions areas signals Each appeared envelopes 8.04-Da difference, non-deuterated deuterated 4C mixture. ratios. discrepancy between less 12% demonstrate possibility 2DE.Fig. 4Measurement abundances reagents. Two (SOD) (for refer I). Spots individual