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Mass spectrometry-based proteomics

Abstract

Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein–protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.

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Figure 1: Generic mass spectrometry (MS)-based proteomics experiment.
Figure 2: Mass spectrometers used in proteome research.
Figure 3: Schematic representation of methods for stable-isotope protein labelling for quantitative proteomics.
Figure 4: Organellar proteomics by combined mass spectrometry (MS) and imaging methods.
Figure 5: Schematic representation of the systems biology paradigm.

References

  1. Pandey, A. & Mann, M. Proteomics to study genes and genomes. Nature 405, 837–846 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. & Whitehouse, C. M. Electrospray ionization for the mass spectrometry of large biomolecules. Science 246, 64–71 (1989).

    ADS  CAS  PubMed  Google Scholar 

  3. Karas, M. & Hillenkamp, F. Laser desorption ionization of proteins with molecular mass exceeding 10000 daltons. Anal. Chem. 60, 2299–2301 (1988).

    CAS  PubMed  Google Scholar 

  4. Aebersold, R. & Goodlett, D. R. Mass spectrometry in proteomics. Chem. Rev. 101, 269–295 (2001).

    CAS  PubMed  Google Scholar 

  5. Mann, M., Hendrickson, R. C. & Pandey, A. Analysis of proteins and proteomes by mass spectrometry. Annu. Rev. Biochem. 70, 437–473 (2001).

    CAS  PubMed  Google Scholar 

  6. Hager, J. W. A new linear ion trap mass spectrometer. Rapid Commun. Mass. Spectrom. 16, 512–526 (2002).

    ADS  CAS  Google Scholar 

  7. Schwartz, J. C., Senko, M. W. & Syka, J. E. A two-dimensional quadrupole ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 13, 659–669 (2002).

    CAS  PubMed  Google Scholar 

  8. Marshall, A. G., Hendrickson, C. L. & Jackson, G. S. Fourier transform ion cyclotron resonance mass spectrometry: a primer. Mass Spectrom. Rev. 17, 1–35 (1998).

    ADS  CAS  PubMed  Google Scholar 

  9. Valaskovic, G. A., Kelleher, N. L. & McLafferty, F. W. Attomole protein characterization by capillary electrophoresis-mass spectrometry. Science 273, 1199–2202 (1996).

    ADS  CAS  PubMed  Google Scholar 

  10. Martin, S. E., Shabanowitz, J., Hunt, D. F. & Marto, J. A. Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 72, 4266–4274 (2000).

    CAS  PubMed  Google Scholar 

  11. Lipton, M. S. et al. Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags. Proc. Natl Acad. Sci. USA 99, 11049–11054 (2002).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Krutchinsky, A. N., Kalkum, M. & Chait, B. T. Automatic identification of proteins with a MALDI-quadrupole ion trap mass spectrometer. Anal. Chem. 73, 5066–5077 (2001).

    CAS  PubMed  Google Scholar 

  13. Medzihradszky, K. F. et al. The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer. Anal. Chem. 72, 552–558 (2000).

    CAS  PubMed  Google Scholar 

  14. Loboda, A. V., Krutchinsky, A. N., Bromirski, M., Ens, W. & Standing, K. G. A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance. Rapid Commun. Mass Spectrom. 14, 1047–1057 (2000).

    ADS  CAS  PubMed  Google Scholar 

  15. Mann, M. & Wilm, M. S. Error tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390–4399 (1994).

    CAS  PubMed  Google Scholar 

  16. Eng, J. K., McCormack, A. L. & Yates, J. R. I An approach to correlate MS/MS data to amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    CAS  PubMed  Google Scholar 

  17. Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Anderson, N. L., Hofmann, J. P., Gemmell, A. & Taylor, J. Global approaches to quantitative analysis of gene-expression patterns observed by use of two-dimensional gel electrophoresis. Clin. Chem. 30, 2031–2036 (1984).

    CAS  PubMed  Google Scholar 

  19. Gygi, S. P., Corthals, G. L., Zhang, Y., Rochon, Y. & Aebersold, R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc. Natl Acad. Sci. USA 97, 9390–9395 (2000).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rabilloud, T. Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2, 3–10 (2002).

    CAS  PubMed  Google Scholar 

  21. Unlu, M., Morgan, M. E. & Minden, J. S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077 (1997).

    CAS  PubMed  Google Scholar 

  22. Gauss, C., Kalkum, M., Lowe, M., Lehrach, H. & Klose, J. Analysis of the mouse proteome. (I) Brain proteins: separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 20, 575–600 (1999).

    CAS  PubMed  Google Scholar 

  23. Hunt, D. F. et al. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255, 1261–1263 (1992).

    ADS  CAS  PubMed  Google Scholar 

  24. Wolters, D. A., Washburn, M. P. & Yates, J. R. III An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73, 5683–5690 (2001).

    CAS  PubMed  Google Scholar 

  25. Link, A. J. et al. Direct analysis of protein complexes using mass spectrometry. Nature Biotechnol. 17, 676–682 (1999).

    CAS  Google Scholar 

  26. Han, D. K., Eng, J., Zhou, H. & Aebersold, R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nature Biotechnol. 19, 946–951 (2001).

    CAS  Google Scholar 

  27. Gygi, S. P., Rist, B., Griffin, T. J., Eng, J. & Aebersold, R. Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags. J. Proteome Res. 1, 47–54 (2002).

    CAS  PubMed  Google Scholar 

  28. Washburn, M. P., Wolters, D. & Yates, J. R. III Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnol. 19, 242–247 (2001).

    CAS  Google Scholar 

  29. Conrads, T. P., Issaq, H. J. & Veenstra, T. D. New tools for quantitative phosphoproteome analysis. Biochem. Biophys. Res. Commun. 290, 885–890 (2002).

    CAS  PubMed  Google Scholar 

  30. Mirgorodskaya, O. A. et al. Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using 18O-labeled internal standards. Rapid Commun. Mass Spectrom. 14, 1226–1232 (2000).

    ADS  CAS  PubMed  Google Scholar 

  31. Yao, X., Freas, A., Ramirez, J., Demirev, P. A. & Fenselau, C. Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Anal. Chem. 73, 2836–2842 (2001).

    CAS  PubMed  Google Scholar 

  32. Gygi, S. P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotechnol. 17, 994–999 (1999).

    CAS  Google Scholar 

  33. Zhou, H., Ranish, J. A., Watts, J. D. & Aebersold, R. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nature Biotechnol. 20, 512–515 (2002).

    CAS  Google Scholar 

  34. Munchbach, M., Quadroni, M., Miotto, G. & James, P. Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety. Anal. Chem. 72, 4047–4057 (2000).

    CAS  PubMed  Google Scholar 

  35. Liu, Y., Patricelli, M. P. & Cravatt, B. F. Activity-based protein profiling: the serine hydrolases. Proc. Natl Acad. Sci. USA 96, 14694–14699 (1999).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Greenbaum, D., Medzihradszky, K. F., Burlingame, A. & Bogyo, M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 7, 569–581 (2000).

    CAS  PubMed  Google Scholar 

  37. Zhou, H., Watts, J. D. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nature Biotechnol. 19, 375–378 (2001).

    CAS  Google Scholar 

  38. Oda, Y., Nagasu, T. & Chait, B. T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nature Biotechnol. 19, 379–382 (2001).

    CAS  Google Scholar 

  39. Zhang, H., Li, X.-J., Martin, D. & Aebersold, R. Quantitative analysis of glycoproteins: applications to serum and membrane proteins. (submitted).

  40. Ong, S. E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    CAS  PubMed  Google Scholar 

  41. Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002).

    CAS  PubMed  Google Scholar 

  42. Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J. & Gygi, S. P. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J. Proteome Res. DOI: 10.1021/pr025556v (2002).

  43. Oshiro, G. et al. Parallel identification of new genes in Saccharomyces cerevisiae. Genome Res. 12, 1210–1220 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Kuster, B., Mortensen, P., Andersen, J. S. & Mann, M. Mass spectrometry allows direct identification of proteins in large genomes. Proteomics 1, 641–650 (2001).

    CAS  PubMed  Google Scholar 

  45. Andersen, J. S. et al. Directed proteomic analysis of the human nucleolus. Curr. Biol. 12, 1–11 (2002).

    PubMed  Google Scholar 

  46. Anderson, N. L. & Anderson, N. G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 1, 845–867 (2002).

    CAS  PubMed  Google Scholar 

  47. Adkins, J. N. et al. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics DOI: 10.1074/mcp.M200066-MCP200 (2002).

  48. Lasonder, E. et al. Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 419, 537–542 (2002).

    ADS  CAS  PubMed  Google Scholar 

  49. Florens, L. et al. A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520–526 (2002).

    ADS  CAS  PubMed  Google Scholar 

  50. Shiio, Y. et al. Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 21, 5088–5096 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Griffin, T. J. et al. Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae. Mol. Cell. Proteomics 1, 323–333 (2002).

    CAS  PubMed  Google Scholar 

  52. Baliga, N. S. et al. Coordinate regulation of energy transduction modules in Halobacterium sp. analyzed by a global systems approach. Proc. Natl Acad. Sci. USA 99, 14913–14918 (2002).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ashman, K., Moran, M. F., Sicheri, F., Pawson, T. & Tyers, M. Cell signalling—the proteomics of it all. Science's STKEhttp://stke.sciencemag.org/cgi/content/full/sigtrans;2001/103/pe33〉 (2001).

  54. Rappsilber, J., Siniossoglou, S., Hurt, E. C. & Mann, M. A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry. Anal. Chem. 72, 267–275 (2000).

    CAS  PubMed  Google Scholar 

  55. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnol. 17, 1030–1032 (1999).

    CAS  Google Scholar 

  56. Gavin, A. C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002).

    ADS  CAS  PubMed  Google Scholar 

  57. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    ADS  CAS  PubMed  Google Scholar 

  58. von Mering, C. et al. Comparative assessment of large-scale data sets of protein–protein interactions. Nature 417, 399–403 (2002).

    ADS  CAS  PubMed  Google Scholar 

  59. Shevchenko, A., Schaft, D., Roguev, A., Pijnappel, W. W. & Stewart, A. F. Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry: analytical perspective. Mol. Cell. Proteomics 1, 204–212 (2002).

    CAS  PubMed  Google Scholar 

  60. Blagoev, B. et al. A proteomics strategy to elucidate functional protein–protein interactions applied to EGF signaling. Nature Biotechnol. advance online publication, 10 February 2003 (doi:10.1038/nbt790).

  61. Ranish, J. A. et al. The study of macromolecular complexes by quantitative proteomics. Nature Genet. (in the press).

  62. MacDonald, J. A., Mackey, A. J., Pearson, W. R. & Haystead, T. A. A strategy for the rapid identification of phosphorylation sites in the phosphoproteome. Mol. Cell. Proteomics 1, 314–322 (2002).

    CAS  PubMed  Google Scholar 

  63. Neubauer, G. et al. Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry. Proc. Natl Acad. Sci. USA 94, 385–390 (1997).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  64. Neubauer, G. et al. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nature Genet. 20, 46–50 (1998).

    CAS  PubMed  Google Scholar 

  65. Rout, M. P. et al. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Rappsilber, J., Ryder, U., Lamond, A. I. & Mann, M. Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231–1245 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Zhou, Z., Licklider, L. J., Gygi, S. P. & Reed, R. Comprehensive proteomic analysis of the human spliceosome. Nature 419, 182–185 (2002).

    ADS  CAS  PubMed  Google Scholar 

  68. Taylor, S. W., Fahy, E. & Ghosh, S. S. Global organellar proteomics. Trends Biotechnol. 21, 82–88 (2003).

    CAS  PubMed  Google Scholar 

  69. Leung, A. K. & Lamond, A. I. In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles. J. Cell Biol. 157, 615–629 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Mann, M. et al. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol. 20, 261–268 (2002).

    CAS  PubMed  Google Scholar 

  71. Mann, M. & Jensen, O. N. Proteomic analysis of post-translational modifications. Nature Biotechnol. (in the press).

  72. MacCoss, M. J. et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc. Natl Acad. Sci. USA 99, 7900–7905 (2002).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  73. Pandey, A. et al. Analysis of receptor signaling pathways by mass spectrometry: identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl Acad. Sci. USA 97, 179–184 (2000).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  74. Steen, H., Kuster, B., Fernandez, M., Pandey, A. & Mann, M. Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway. J. Biol. Chem. 277, 1031–1039 (2002).

    CAS  PubMed  Google Scholar 

  75. Ficarro, S. B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nature Biotechnol. 20, 301–305 (2002).

    CAS  Google Scholar 

  76. Peng, J. & Gygi, S. P. Proteomics: the move to mixtures. J. Mass Spectrom. 36, 1083–1091 (2001).

    ADS  CAS  PubMed  Google Scholar 

  77. Hanson, C. L., Fucini, P., Ilag, L. L., Nierhaus, K. H. & Robinson, C. V. Dissociation of intact Escherichia coli ribosomes in a mass spectrometer—evidence for conformational change in a ribosome elongation factor G complex. J. Biol. Chem. 278, 1259–1267 (2002).

    PubMed  Google Scholar 

  78. Oh, H. et al. Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy. Proc. Natl Acad. Sci. USA 99, 15863–15868 (2002).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  79. Cohen, S. L. & Chait, B. T. Mass spectrometry as a tool for protein crystallography. Annu. Rev. Biophys. Biomol. Struct. 30, 67–85 (2001).

    CAS  PubMed  Google Scholar 

  80. Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 95, 14863–14868 (1998).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  81. Aebersold, R. & Watts, J. D. The need for national centers for proteomics. Nature Biotechnol. 20, 651 (2002).

    CAS  Google Scholar 

  82. Mann, M. A home for proteomics data? Nature 420, 21 (2002).

    ADS  CAS  Google Scholar 

  83. Petricoin, E. F. et al. Use of proteomic patterns in serum to identify ovarian cancer. Lancet 359, 572–577 (2002).

    CAS  PubMed  Google Scholar 

  84. Mørtz, E. et al. Sequence tag identification of intact proteins by matching tandem mass spectral data against sequence data bases. Proc. Natl Acad. Sci. USA 93, 8264–8267 (1996).

    ADS  PubMed  PubMed Central  Google Scholar 

  85. Stoeckli, M., Chaurand, P., Hallahan, D. E. & Caprioli, R. M. Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nature Med. 7, 493–496 (2001).

    CAS  PubMed  Google Scholar 

  86. Goodlett, D. R. et al. Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching. Anal. Chem. 72, 1112–1118 (2000).

    CAS  PubMed  Google Scholar 

  87. Smith, R. D. et al. An accurate mass tag strategy for quantitative and high-throughput proteome measurements. Proteomics 2, 513–523 (2002).

    CAS  PubMed  Google Scholar 

  88. Ideker, T. et al. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 292, 929–934 (2001).

    ADS  CAS  PubMed  Google Scholar 

  89. Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 43, 717–731 (2002).

    CAS  PubMed  Google Scholar 

  90. Guina, T. et al. Quantitative proteomic analysis of Pseudomonas aeruginosa indicates synthesis of quinolone signal in adaptation to cystic fibrosis airways. Proc. Natl Acad. Sci. USA (in the press).

  91. Fox, A. H. et al. Paraspeckles. A novel nuclear domain. Curr. Biol. 12, 13–25 (2002).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank members of the Institute of Systems Biology (ISB) and the Center for Experimental BioInformatics (CEBI) for critical reading of the manuscript, preparation of figures and fruitful discussions, especially L. Foster, S.-E. Ong, J. Andersen and L. Feltz. CEBI is supported by a grant from the Danish Natural Research Foundation. R.A. is supported by grants from the National Institute of Health and Oxford GlycoSciences, and a contract from the National Heart, Lung, and Blood Institute, National Institutes of Health. The ISB is supported in part by a gift from Merck and Co.

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Aebersold, R., Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003). https://doi.org/10.1038/nature01511

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