Advances in Proteome Analysis by Mass Spectrometry (2001)

two-dimensional electrophoresis reverse-phase microcapillary liquid chromatography mass spectrometry tandem mass spectrometry electrospray ionization matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation isotope-coded affinity tag strong cation exchange high performance liquid chromatography The interpretation of the information contained in the genomic sequence of a species with respect to the structure, function, and control of biological processes is a technical and conceptual challenge for current research methods. Systematic and quantitative analysis of gene expression is emerging as a valuable tool to diagnostically distinguish between cell types (1Golub T.R. Slonim D.K. Tamayo P. Huard C. Gaasenbeek M. Mesirov J.P. Coller H. Loh M.L. Downing J.R. Caligiuri M.A. Bloomfield C.D. Lander E.S. Science. 1999; 286: 531-537Crossref PubMed Scopus (9190) Google Scholar, 2Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T., Yu, X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson Jr., J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Staudt L.M. et al.Nature. 2000; 403: 503-511Crossref PubMed Scopus (7996) Google Scholar, 3Scherf U. Ross D.T. Waltham M. Smith L.H. Lee J.K. Tanabe L. Kohn K.W. Reinhold W.C. Myers T.G. Andrews D.T. Scudiero D.A. Eisen M.B. Sausville E.A. Pommier Y. Botstein D. Brown P.O. Weinstein J.N. Nat. Genet. 2000; 24: 236-244Crossref PubMed Scopus (1275) Google Scholar, 4Ross D.T. Scherf U. Eisen M.B. Perou C.M. Rees C. Spellman P. Iyer V. Jeffrey S.S. Van de Rijn M. Waltham M. Pergamenschikov A. Lee J.C. Lashkari D. Shalon D. Myers T.G. Weinstein J.N. Botstein D. Brown P.O. Nat. Genet. 2000; 24: 227-235Crossref PubMed Scopus (1821) Google Scholar, 5Lockhart D.J. Winzeler E.A. Nature. 2000; 405: 827-836Crossref PubMed Scopus (1691) Google Scholar) and to differentiate between states (metabolic, activation, pathological) of a particular cell type (5Lockhart D.J. Winzeler E.A. Nature. 2000; 405: 827-836Crossref PubMed Scopus (1691) Google Scholar, 6Lashkari D.A. DeRisi J.L. McCusker J.H. Namath A.F. Gentile C. Hwang S.Y. Brown P.O. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13057-13062Crossref PubMed Scopus (530) Google Scholar, 7Marton M.J. DeRisi J.L. Bennett H.A. Iyer V.R. Meyer M.R. Roberts C.J. Stoughton R. Burchard J. Slade D. Dai H. Bassett Jr., D.E. Hartwell L.H. Brown P.O. Friend S.H. Nat. Med. 1998; 4: 1293-1301Crossref PubMed Scopus (592) Google Scholar). More elaborate strategies such as the combination of systematic, quantitative gene expression analysis with targeted, hypothesis-guided perturbations of cells are being explored for the comprehensive mechanistic analysis of cellular pathways and processes (5Lockhart D.J. Winzeler E.A. Nature. 2000; 405: 827-836Crossref PubMed Scopus (1691) Google Scholar, 7Marton M.J. DeRisi J.L. Bennett H.A. Iyer V.R. Meyer M.R. Roberts C.J. Stoughton R. Burchard J. Slade D. Dai H. Bassett Jr., D.E. Hartwell L.H. Brown P.O. Friend S.H. Nat. Med. 1998; 4: 1293-1301Crossref PubMed Scopus (592) Google Scholar, 8Holstege F.C. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 9Roberts C.J. Nelson B. Marton M.J. Stoughton R. Meyer M.R. Bennett H.A. He Y.D. Dai H. Walker W.L. Hughes T.R. Tyers M. Boone C. Friend S.H. Science. 2000; 287: 873-880Crossref PubMed Scopus (719) Google Scholar, 10Ideker T. Thorsson V. Ranish J.A. Christmas R. Buhler J. Eng J.K. Bumgarner R. Goodlett D.R. Aebersold R. Hood L. Science. 2001; 292: 929-934Crossref PubMed Scopus (1649) Google Scholar). Measuring gene expression at the protein level is potentially more informative than the corresponding measurement at the mRNA level. Proteins, the major catalysts of biological function, contain several dimensions of information that collectively indicate the actual rather than the potential functional state as indicated by mRNA analysis. These include the abundance, state of modification, subcellular location, and three-dimensional structure of proteins and their association with each other and/or with biomolecules of different types. Mass spectrometry has become the analytical technology of choice for many of the aspects of proteome analyses that are reflected by the covalent structure of proteins. This review describes recent, innovative advances in mass spectrometry-based proteome analysis that collectively have converged into a generic new approach to proteomics. The specific advances include high throughput protein identification by multidimensional chromatography, automated tandem mass spectrometry and sequence data base searching, accurate quantification by the application of stable isotope dilution theory to protein analysis, and the targeted isolation of selected analytes by the use of highly selective chemistries. Selected applications of these methods along with speculations about future prospects and directions of proteomics research are also included. The development of methods to separate complex protein mixtures at high resolution by two-dimensional gel electrophoresis (2DE)1 into reproducible patterns (11O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar, 12Klose J. Humangenetik. 1975; 26: 231-243PubMed Google Scholar) presented the opportunity to diagnose quantitative and qualitative differences in the protein composition of two or more cell or tissue samples long before gene array techniques to measure gene expression were conceived. Unfortunately, 2DE by itself was an essentially descriptive technique and, without the availability of reliable tools for the identification of the separated protein species, of limited utility as a molecular biology research tool. This changed in the early 1990s when two revolutionary techniques, matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) (13Karas M. Hillenkamp F. Anal. Chem. 1988; 60: 2299-2301Crossref PubMed Scopus (4828) Google Scholar, 14Hillenkamp F. Karas M. Beavis R.C. Chait B.T. Anal. Chem. 1991; 63: 1193A-1203ACrossref PubMed Google Scholar) and electrospray ionization (ESI) (15Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Science. 1989; 246: 64-71Crossref PubMed Scopus (6314) Google Scholar, 16Banks Jr., J.F. Whitehouse C.M. Methods Enzymol. 1996; 270: 486-519Crossref PubMed Google Scholar) MS and tandem mass spectrometry (MS/MS) replaced the slower and less sensitive chemical degradation methods (17Aebersold R.H. Leavitt J. Saavedra R.A. Hood L.E. Kent S.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6970-6974Crossref PubMed Scopus (630) Google Scholar, 18Lin C.S. Aebersold R.H. Kent S.B. Varma M. Leavitt J. Mol. Cell. Biol. 1988; 8: 4659-4668Crossref PubMed Scopus (106) Google Scholar) as the methods of choice for the identification of proteins separated by 2DE (19Humphery-Smith I. Cordwell S.J. Blackstock W.P. Electrophoresis. 1997; 18: 1217-1242Crossref PubMed Scopus (229) Google Scholar, 20Dunn M.J. Corbett J.M. Methods Enzymol. 1996; 271: 177-203Crossref PubMed Google Scholar, 21Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Boucherie H. Mann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Crossref PubMed Scopus (1298) Google Scholar, 22Clauser K.R. Hall S.C. Smith D.M. Webb J.W. Andrews L.E. Tran H.M. Epstein L.B. Burlingame A.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5072-5076Crossref PubMed Scopus (201) Google Scholar, 23Pandey A. Mann M. Nature. 2000; 405: 837-846Crossref PubMed Scopus (1942) Google Scholar, 24Patterson S.D. Aebersold R. Electrophoresis. 1995; 16: 1791-1814Crossref PubMed Scopus (368) Google Scholar). Typically, these methods involved excision of gel bands of interest, in-gel digestion of the proteins contained in the band using the enzyme trypsin (25Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7806) Google Scholar), and finally mass spectrometric analysis of the peptides produced. Protein identification was accomplished using peptide-mass fingerprinting by MALDI-TOF MS, as initially described by Henzelet al. (26Henzel W.J. Billeci T.M. Stults J.T. Wong S.C. Grimley C. Watanabe C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5011-5015Crossref PubMed Scopus (1181) Google Scholar) and independently by others (reviewed in Ref.23Pandey A. Mann M. Nature. 2000; 405: 837-846Crossref PubMed Scopus (1942) Google Scholar), nano-ESI tandem mass spectrometry (MS/MS) (27Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar, 28Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1703) Google Scholar), or reverse-phase (RP) microcapillary liquid chromatography (μLC) ESI MS/MS (29Shabanowitz J. Settlage R.E. Marto J.A. Christian R.E. White F.M. Russo P.S. Martin S.E. Hunt D.F. Burlingame A.L. Carr S.A. Baldwin M.A. Mass Spectrometry in Biology and Medicine. Humana Press, Totowa, NJ2000: 163-177Google Scholar, 30Hunt D.F. Henderson R.A. Shabanowitz J. Sakaguchi K. Michel H. Sevilir N. Cox A.L. Appella E. Engelhard V.H. Science. 1992; 255: 1261-1263Crossref PubMed Scopus (1049) Google Scholar, 31McCormack A.L. Schieltz D.M. Goode B. Yang S. Barnes G. Drubin D. Yates III, J.R. Anal. Chem. 1997; 69: 767-776Crossref PubMed Scopus (446) Google Scholar, 32Davis M.T. Stahl D.C. Hefta S.A. Lee T.D. Anal. Chem. 1995; 67: 4549-4556Crossref PubMed Scopus (201) Google Scholar, 33Ducret A. Van Oostveen I. Eng J.K. Yates III, J.R. Aebersold R. Protein Sci. 1998; 7: 706-719Crossref PubMed Scopus (280) Google Scholar, 34Haynes P.A. Fripp N. Aebersold R. Electrophoresis. 1998; 19: 939-945Crossref PubMed Scopus (62) Google Scholar) using automated, data-dependent scanning and dynamic exclusion of peptide ions already analyzed in the same experiment (29Shabanowitz J. Settlage R.E. Marto J.A. Christian R.E. White F.M. Russo P.S. Martin S.E. Hunt D.F. Burlingame A.L. Carr S.A. Baldwin M.A. Mass Spectrometry in Biology and Medicine. Humana Press, Totowa, NJ2000: 163-177Google Scholar, 35Gatlin C.L. Eng J.K. Cross S.T. Detter J.C. Yates III, J.R. Anal. Chem. 2000; 72: 757-763Crossref PubMed Scopus (215) Google Scholar, 36Figeys D. Aebersold R. Electrophoresis. 1997; 18: 360-368Crossref PubMed Scopus (72) Google Scholar, 37Courchesne P.L. Jones M.D. Robinson J.H. Spahr C.S. McCracken S. Bentley D.L. Luethy R. Patterson S.D. Electrophoresis. 1998; 19: 956-967Crossref PubMed Scopus (39) Google Scholar). The tandem mass spectra produced by the collision-induced dissociation (CID) (38Hunt D.F. Yates III, J.R. Shabanowitz J. Winston S. Hauer C.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6233-6237Crossref PubMed Scopus (1074) Google Scholar, 39Papayannopoupos I.A. Mass Spectrom. Rev. 1995; 14: 49-73Crossref Scopus (587) Google Scholar) of selected peptides are searched against theoretical tandem mass spectra of peptide sequences contained in a data base using a variety of search algorithms (40Beavis R.C. Fenyo D. Mann M. Blackstock W. Proteomics: a Trends Guide. Elsevier, London2000: 22-27Google Scholar), the pioneering program being Sequest developed by Eng et al.(41Eng J. McCormack A.L. Yates III, J.R. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5419) Google Scholar). The specific techniques and instruments have been reviewed in detail and are not discussed further here (42Blackstock W. Mann M. Blackstock W. Proteomics: a Trends Guide. Elsevier, London2000: 12-17Google Scholar, 43Yates III, J.R. Trends Genet. 2000; 16: 5-8Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). It has become apparent that the 2DE-MS method as most frequently practiced has significant, inherent limitations. First, the combination of limited sample capacity and limited detection sensitivity of 2DE restricts the detection of low abundance proteins. If total yeast cell lysates are separated and detected by silver staining, proteins present at less than 1000 copies per cell are not detected (44Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9390-9395Crossref PubMed Scopus (1213) Google Scholar). As proteins expressed at low abundance may make up a large portion of a given proteome (44Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9390-9395Crossref PubMed Scopus (1213) Google Scholar), it is apparent that the proteins detected by 2DE do not give a true representation of all the expressed proteins. Second, despite substantial recent advances (45Rabilloud T. Adessi C. Giraudel A. Lunardi J. Electrophoresis. 1997; 18: 307-316Crossref PubMed Scopus (408) Google Scholar, 46Gorg A. Obermaier C. Boguth G. Harder A. Scheibe B. Wildgruber R. Weiss W. Electrophoresis. 2000; 21: 1037-1053Crossref PubMed Scopus (1638) Google Scholar), the separation of transmembrane proteins by 2DE remains challenging. Third, a substantial fraction of spots contain more than one protein and/or differentially modified or processed forms of a protein that migrate to different positions in the gel, thus complicating quantification (44Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9390-9395Crossref PubMed Scopus (1213) Google Scholar). Fourth, the method is based on the sequential identification of individually processed protein spots, which limits its throughput. Fifth, the method is inherently labor-intensive and requires a high skill level, which limits the potential for full automation. Collectively, these limitations indicate the need for the development of improved or alternative technologies if routine proteome analysis is to become a reality. To address these limitations, incremental improvements of the 2DE-MS approach have been made that include sample prefractionation prior to 2DE (47Corthals G.L. Molloy M.P. Herbert B.R. Williams K.L. Gooley A.A. Electrophoresis. 1997; 18: 317-323Crossref PubMed Scopus (83) Google Scholar), the use of fluorescent protein dyes with enhanced detection sensitivity (48Steinberg T.H. Chernokalskaya E. Berggren K. Lopez M.F. Diwu Z. Haugland R.P. Patton W.F. Electrophoresis. 2000; 21: 486-496Crossref PubMed Scopus (75) Google Scholar), and the use of gels with expanded separation range (zoom gels) (46Gorg A. Obermaier C. Boguth G. Harder A. Scheibe B. Wildgruber R. Weiss W. Electrophoresis. 2000; 21: 1037-1053Crossref PubMed Scopus (1638) Google Scholar, 49Wildgruber R. Harder A. Obermaier C. Boguth G. Weiss W. Fey S.J. Larsen P.M. Gorg A. Electrophoresis. 2000; 21: 2610-2616Crossref PubMed Scopus (161) Google Scholar) to improve the detection sensitivity of low abundance proteins, the search for new detergent systems to maximize solubility of membrane proteins (45Rabilloud T. Adessi C. Giraudel A. Lunardi J. Electrophoresis. 1997; 18: 307-316Crossref PubMed Scopus (408) Google Scholar), and the development of robotic and software systems to increase the level of automation of the process (42Blackstock W. Mann M. Blackstock W. Proteomics: a Trends Guide. Elsevier, London2000: 12-17Google Scholar, 50Lopez M.F. Electrophoresis. 2000; 21: 1082-1093Crossref PubMed Scopus (118) Google Scholar). Concurrently, an alternative technique has been emerging that has the potential to systematically identify and quantify all the proteins in a cell or tissue type. It is based on three principles. The first is rapid protein identification by automated tandem mass spectrometry and sequence data base searching. Essentially the same methods developed for the identification of gel-separated proteins are applied to identify the components of unseparated protein mixtures. The second is the determination of the ratio of abundance (relative quantification) for proteins present in different protein samples by stable isotope dilution. Stable isotope dilution theory (51De Leenheer A.P. Thienpont L.M. Mass Spectrom. Rev. 1992; 11: 249-307Crossref Scopus (97) Google Scholar) states that the relative signal intensity in a mass spectrometer of two analytes that are chemically identical but of different stable isotope composition (and thus distinguishable in a mass analyzer) are a true representation of the relative abundance of the two analytes in the sample. The third principle is the targeted isolation of selected peptide analytes from complex peptide mixtures via specific chemical reactions. Collectively, the three components permit the relative measurement of abundance and the identification of the components of very complex protein mixtures rapidly and with a high degree of automation, without the need to separate protein mixtures prior to analysis. Variations of this technology also have the potential to systematically and quantitatively determine properties of proteins that reflect their functional state. These include the phosphorylation state and the activity of some of The and early applications of this second proteomics technology are described The challenge comprehensive proteomics approach is one of and very complex mixtures of proteins in which components in abundance by or more of information to for comprehensive of expressed proteins. To this the combination of selective of proteins with stable affinity and multidimensional liquid chromatography in with automated, data-dependent tandem mass spectrometry and sequence data base has and has been to at some of the limitations of the approach to proteomics. The of proteins at specific in a complex by and selective of the peptide has to an method for the analysis of unseparated protein The contained in the of is a targeted for the and of proteins and peptides G.L. Methods Enzymol. PubMed Scopus Google S. Chait B.T. Anal. Chem. 1998; PubMed Scopus Google Scholar). The of in protein sequences it an to for the of the of peptide mixtures of all peptides in the yeast contain a D. J. Chait B.T. Electrophoresis. 1998; 19: PubMed Scopus Google as of the total proteins in the S. contain at one D. J. Chait B.T. Electrophoresis. 1998; 19: PubMed Scopus Google Scholar), and identification of the peptides the comprehensive identification of expressed proteins, the of the sample is of the of peptide mixtures prior to mass spectrometric analysis is for several First, the of a of the peptides by of a protein the representation of the selected peptides in the sample that is the The of the sample by selective is for and low abundance proteins. Second, as low peptides are automated MS/MS analysis to the long of the the for the identification of a of the Third, the of the that is indicated by the specific between the and the a for sequence data base searching. The use of selective of for the of the peptide samples by of protein mixtures prior to mass spectrometric analysis has been by Spahr et al. C.S. S.A. Robinson J.H. Davis M.T. M.D. G. Patterson S.D. Electrophoresis. 2000; 21: PubMed Scopus Google Scholar). The using a and these peptides were using and by This experiment was of a in which a total of proteins from samples were and of peptides is also the of the isotope-coded affinity tag approach that was developed in S.P. B. S.A. F. Aebersold R. Nat. 1999; PubMed Scopus Google Scholar). this approach the proteins present in two samples the proteins expressed by a cell two different are on the of their using one of two different but chemically identical of the is an on the to as the and the other is an the have been replaced with As the of peptide with and of the are chemically to stable isotope dilution theory (51De Leenheer A.P. Thienpont L.M. Mass Spectrom. Rev. 1992; 11: 249-307Crossref Scopus (97) Google Scholar) as for accurate protein The relative of each protein present in the two biological samples is by the relative signal of of peptides using an mass The identification of the proteins is accomplished by the to MS/MS in which it peptides for for quantitative protein analysis using selective peptide and MS have also been developed M. M. G. P. Anal. Chem. 2000; 72: PubMed Scopus Google Scholar, J. A. M. Zhang X. A. M. F. J. 2000; PubMed Scopus Google Scholar). et al. M. M. G. P. Anal. Chem. 2000; 72: PubMed Scopus Google Scholar) the of peptides from proteins using a stable to the of on protein expression in of these peptides mass that de peptide by MS/MS analysis sequence determination from interpretation of MS/MS spectra without the need for data base approach the of on peptides with or which for relative quantification of protein expression and de alternative approach to quantitative protein analysis of stable into differentially expressed proteins from cell on or or in stable Y. K. Cross D. Chait B.T. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, L. Jensen P. G. M. K. S. N. J. Smith R. J. Am. Chem. Soc. 1999; Scopus Google Scholar). with selective and of proteins, that liquid chromatography in with automated ESI tandem mass spectrometric analysis have been developed as an alternative to 2DE for complex protein mixtures M.P. Yates J.R. Mann M. Blackstock W. Proteomics: a Trends Guide. Elsevier, London2000: Scholar, Lewis J.W. Anal. Chem. 1997; 69: PubMed Scopus (325) Google Scholar, Eng J. Schieltz D.M. E. D.R. Yates J.R. Nat. 1999; PubMed Scopus Google Scholar). et al. Eng J. Schieltz D.M. E. D.R. Yates J.R. Nat. 1999; PubMed Scopus Google Scholar) described an that a two-dimensional with strong cation exchange and sequential separation of peptides first by and by by MS/MS analysis. This method has for the comprehensive analysis of protein Eng J. Schieltz D.M. E. D.R. Yates J.R. Nat. 1999; PubMed Scopus Google Scholar), and more it has been applied to the identification of proteins, low abundance proteins, from a yeast M.P. D. Yates III, J.R. Nat. 2001; 19: PubMed Scopus Google Scholar). et al. (44Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9390-9395Crossref PubMed Scopus (1213) Google Scholar) have a approach that high performance liquid chromatography in with MS/MS and have that this method the analysis of low abundance proteins in S. that are not sensitive to have also this multidimensional with the described to an approach to the quantitative analysis of protein expression have applied this to the of in the protein expression in yeast cells S.P. B. S.A. F. Aebersold R. Nat. 1999; PubMed Scopus Google Scholar) to differentially in the membrane protein composition in cells D.K. Eng J. H. Aebersold R. Nat. 2001; 19: PubMed Scopus Google Scholar), as as to differences in protein from and cell E. and R. in by protein expression data with array data the corresponding mRNA expression have the tools for the analysis of gene expression on a level. an have applied these two gene expression to the analysis of pathways in yeast T. Thorsson V. Ranish J.A. Christmas R. Buhler J. Eng J.K. Bumgarner R. Goodlett D.R. Aebersold R. Hood L. Science. 2001; 292: 929-934Crossref PubMed Scopus (1649) Google Scholar). It that the approach as as second proteomics technologies to rapidly and the These advances by and software to mass is to second proteomics The combination of stable isotope and automated MS/MS also to quantitatively and systematically properties of other than their have developed a method for the quantitative analysis of protein phosphorylation on a H. Aebersold R. Nat. 2001; 19: PubMed Scopus Google Scholar). It the specific of on the isolation of these for mass spectrometric analysis using a This method also a of the of the peptides with stable isotope thus the quantitative analysis of proteins by MS/MS in a to the This approach applied to the proteins contained in a total yeast cell in a MS/MS experiment phosphorylation on many of which were not to proteins. The development of Y. M.P. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, M.P. M.A. 1999; PubMed Scopus Google Scholar, D. Burlingame A. M. Chem. Biol. 2000; 7: Full Text Full Text PDF PubMed Scopus Google Scholar) that proteins based on their state of activity rather than on the of specific an opportunity to Chem. Biol. 2000; 4: PubMed Scopus Google Scholar). This is by the of chemical that to the of specific in an et al. Y. M.P. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) have developed a that the of and et al. D. Burlingame A. M. Chem. Biol. 2000; 7: Full Text Full Text PDF PubMed Scopus Google Scholar) have developed an for the selective of with stable isotope the of the and activity of selected of proteins by It that to the performance of mass detection sample mass and mass to not all on the same The recent of a mass spectrometer that a with a a cell and a A.V. M. W. Mass Spectrom. 2000; 14: PubMed Scopus Google Scholar, A. A. Shevchenko A. W. Anal. Chem. 2000; 72: PubMed Scopus Google Scholar) the to the to automation, and mass of MALDI-TOF MS with the of have the to for the analysis of proteins A. S.P. B. A. W. Aebersold R. Anal. Chem. 2001; PubMed Scopus Google Scholar) with the of the approach being that proteins expression between the two biological by MS/MS analysis, or differences in expression The to the differentially expressed proteins to a increase in throughput for protein expression recent in is the development of a mass spectrometer J.M. Baldwin M.A. P. M.L. Burlingame A.L. Anal. Chem. 2000; 72: PubMed Scopus Google Scholar). This the same of the with the potential for analysis and thus the potential to increase sample the identification of of proteins per type of that potential for is the mass which and mass relative to other mass L. Jensen P. G. M. K. S. N. J. Smith R. J. Am. Chem. Soc. 1999; Scopus Google Scholar, S.E. Shabanowitz J. Hunt D.F. Marto J.A. Anal. Chem. 2000; 72: PubMed Scopus Google Scholar, J. R. Smith Anal. Chem. 1999; PubMed Scopus Google Scholar). It has been in yeast that with the mass by analysis of peptides is to identify the peptides (and thus the proteins from which are from a data base without the need for tandem mass spectrometric analysis D.R. B. L. O. Smith Aebersold R. Anal. Chem. 2000; 72: PubMed Scopus Google Scholar, T.D. L. Smith Anal. Chem. 2000; 72: PubMed Scopus Google Scholar). This for the detection of low abundance proteins that are many when peptides by MS/MS and thus this approach has for improved sensitivity and throughput in Collectively, these advances are to the performance of with respect to level of automation, sample and it that the to measure properties of proteins proteomics to the comprehensive analysis of biological Goodlett for on this