[go: up one dir, main page]
More Web Proxy on the site http://driver.im/
Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Large-scale analysis of the yeast proteome by multidimensional protein identification technology

Nature Biotechnology volume 19, pages 242–247 (2001)Cite this article

Abstract

We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Multidimensional protein identification technology (MudPIT).
Figure 2: Codon adaptation index (CAI) distribution of the identified S. cerevisiae proteome and the predicted S. cerevisiae genome.
Figure 3: Peptide mapping of the integral membrane protein PMA1.
Figure 4: Sensitivity of MudPIT to a wide variety of protein classes.

Similar content being viewed by others

References

  1. Lockhart, D.J. & Winzeler, E.A. Genomics, gene expression and DNA arrays. Nature 405, 827–836 (2000).

    Article  CAS  Google Scholar 

  2. Kawamoto, S., Matsumoto, Y., Mizuno, K., Okubo, K. & Matsubara, K. Expression profiles of active genes in human and mouse livers. Gene 174, 151–158 (1996).

    Article  CAS  Google Scholar 

  3. Anderson, L. & Seilhamer, J. A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18, 533–537 (1997).

    Article  CAS  Google Scholar 

  4. Futcher, B., Latter, G.I., Monardo, P., McLaughlin, C.S. & Garrels, J.I. A sampling of the yeast proteome. Mol. Cell. Biol. 19, 7357–7368 (1999).

    Article  CAS  Google Scholar 

  5. Gygi, S.P., Rochon, Y., Franza, B.R. & Aebersold, R. Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 19, 1720–1730 (1999).

    Article  CAS  Google Scholar 

  6. Hatizmanikatis, V. & Lee, K.H. Dynamical analysis of gene networks requires both mRNA and protein expression information. Metabol. Eng. 1, 275–281 (1999).

    Article  Google Scholar 

  7. Hatzimanikatis, V., Choe, L.H. & Lee, K.H. Proteomics: theoretical and experimental considerations. Biotechnol. Prog. 15, 312–318 (1999).

    Article  CAS  Google Scholar 

  8. Hanash, S.M. Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current status. Electrophoresis 21, 1202–1209 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Washburn, M.P. & Yates, J.R. Analysis of the microbial proteome. Curr. Opin. Microbiol. 3, 292–297 (2000).

    Article  CAS  Google Scholar 

  11. Langen, H. et al. Two-dimensional map of the proteome of Haemophilus influenzae. Electrophoresis 21, 411–429 (2000).

    Article  CAS  Google Scholar 

  12. Oh-Ishi, M., Satoh, M. & Maeda, T. Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins. Electrophoresis 21, 1653–1669 (2000).

    Article  CAS  Google Scholar 

  13. Corthals, G.L., Wasinger, V.C., Hochstrasser, D.F. & Sanchez, J.C. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 21, 1104–1115 (2000).

    Article  CAS  Google Scholar 

  14. Fountoulakis, M., Takacs, M.F., Berndt, P., Langen, H. & Takacs, B. Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography. Electrophoresis 20, 2181–2195 (1999).

    Article  CAS  Google Scholar 

  15. Fountoulakis, M., Takacs, M.F. & Takacs, B. Enrichment of low-copy-number gene products by hydrophobic interaction chromatography. J. Chromatogr. A 833, 157–168 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Molloy, M.P. Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal. Biochem. 280, 1–10 (2000).

    Article  CAS  Google Scholar 

  18. Santoni, V., Molloy, M. & Rabilloud, T. Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–70 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. McCormack, A.L. et al. Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal. Chem. 69, 767–776 (1997).

    Article  CAS  Google Scholar 

  21. Eng, J.K., McCormack, A.L. & Yates, J.R.I. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    Article  CAS  Google Scholar 

  22. Giddings, J.C. Concepts and comparisons in multidimensional chromatography. J. High Res. Chromatogr. 10, 319–323 (1987).

    Article  CAS  Google Scholar 

  23. Washburn, M.P. & Yates, J.R. Novel methods of proteome analysis: multidimensional chromatography and mass spectrometry. Proteomics: A Current Trends Supplement, 28–32 (2000).

  24. Mewes, H.W. et al. MIPS: a database for genomes and protein sequences. Nucleic Acids Res. 28, 37–40 (2000).

    Article  CAS  Google Scholar 

  25. Sharp, P.M. & Li, W.H. The Codon Adaptation Index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 15, 1281–1295 (1987).

    Article  CAS  Google Scholar 

  26. Peterson, C.L. & Workman, J.L. Promoter targeting and chromatin remodeling by the SWI/SNF complex. Curr. Opin. Genet. Dev. 10, 187–192 (2000).

    Article  CAS  Google Scholar 

  27. Cairns, B.R., Kim, Y.J., Sayre, M.H., Laurent, B.C. & Kornberg, R.D. A multisubunit complex containing the SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products isolated from yeast. Proc. Natl. Acad. Sci. USA 91, 1950–1954 (1994).

    Article  CAS  Google Scholar 

  28. Culotta, V.C. et al. The copper chaperone for superoxide dismutase. J. Biol. Chem. 272, 23469–23472 (1997).

    Article  CAS  Google Scholar 

  29. Liu, Q. et al. Site-directed mutagenesis of the yeast V-ATPase A subunit. J. Biol. Chem. 272, 11750–11756 (1997).

    Article  CAS  Google Scholar 

  30. Lee, B.N. & Elion, E.A. The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components. Proc. Natl. Acad. Sci. USA 96, 12679–12684 (1999).

    Article  CAS  Google Scholar 

  31. Sprague, G.F. Jr., Control of MAP kinase signaling specificity or how not to go HOG wild. Genes Dev. 12, 2817–2820 (1998).

    Article  CAS  Google Scholar 

  32. Perrot, M. et al. Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999). Electrophoresis 20, 2280–2298 (1999).

    Article  CAS  Google Scholar 

  33. Costanzo, M.C. et al. The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive resources for the organization and comparison of model organism protein information. Nucleic Acids Res. 28, 73–76 (2000).

    Article  CAS  Google Scholar 

  34. Klein, P., Kanehisa, M. & DeLisi, C. The detection and classification of membrane-spanning proteins. Biochim. Biophys. Acta 815, 468–476 (1985).

    Article  CAS  Google Scholar 

  35. Goffeau, A., Nakai, K., Slonimski, P., Risler, J.L. & Slominski, P. The membrane proteins encoded by yeast chromosome III genes. FEBS Lett. 325, 112–117 (1993).

    Article  CAS  Google Scholar 

  36. Ambesi, A., Miranda, M., Petrov, V.V. & Slayman, C.W. Biogenesis and function of the yeast plasma-membrane H(+)-ATPase. J. Exp. Biol. 203, 155–160 (2000).

    CAS  PubMed  Google Scholar 

  37. Auer, M., Scarborough, G.A. & Kuhlbrandt, W. Three-dimensional map of the plasma membrane H+-ATPase in the open conformation. Nature 392, 840–843 (1998).

    Article  CAS  Google Scholar 

  38. Zhang, P., Toyoshima, C., Yonekura, K., Green, N.M. & Stokes, D.L. Structure of the calcium pump from sarcoplasmic reticulum at 8-Å resolution. Nature 392, 835–839 (1998).

    Article  CAS  Google Scholar 

  39. Kuhlbrandt, W., Auer, M. & Scarborough, G.A. Structure of the P-type ATPases. Curr. Opin. Struct. Biol. 8, 510–516 (1998).

    Article  CAS  Google Scholar 

  40. McIntosh, D.B. Portrait of a P-type pump. Nat. Struct. Biol. 7, 532–535 (2000).

    Article  CAS  Google Scholar 

  41. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405, 647–655 (2000).

    Article  CAS  Google Scholar 

  42. Shevchenko, A. et al. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. USA 93, 14440–14445 (1996).

    Article  CAS  Google Scholar 

  43. Garrels, J.I. et al. Proteome studies of Saccharomyces cerevisiae: identification and characterization of abundant proteins. Electrophoresis 18, 1347–1360 (1997).

    Article  CAS  Google Scholar 

  44. Nilsson, C.L. & Davidsson P. New separation tools for comprehensive studies of protein expression by mass spectrometry. Mass Spectrom. Rev. 19, 390–397 (2000).

    Article  CAS  Google Scholar 

  45. Molloy, M.P. et al. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267, 2871–2881 (2000).

    Article  CAS  Google Scholar 

  46. Pasa-Tolic, L. et al. High throughput proteome-wide precision measurements of protein expression using mass spectrometry. J. Am. Chem. Soc. 121, 7949–7950 (1999).

    Article  CAS  Google Scholar 

  47. Oda, Y., Huang, K., Cross, F.R., Cowburn, D. & Chait, B.T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591–6596 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  49. Münchbach, 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).

    Article  Google Scholar 

  50. Jones, E.W. Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol. 194, 428–453 (1991).

    Article  CAS  Google Scholar 

  51. Gatlin, C.L., Kleemann, G.R., Hays, L.G., Link, A.J. & Yates, J.R. Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem. 263, 93–101 (1998).

    Article  CAS  Google Scholar 

  52. Aitken, A., Geisow, M.J., Findlay, J.B.C., Holmes, C. & Yarwood, A. Peptide preparation and characterization. In Protein sequencing: a practical approach (eds Findlay, J.B.C. & Geisow, M.J.) 43–68 (IRL Press, New York, NY; 1989).

    Google Scholar 

Download references

Acknowledgements

The authors thank Jimmy Eng, David Schieltz, David Tabb, and Laurence Florens for valuable discussions during the preparation of this manuscript. The authors acknowledge funding from the National Institutes of Health R33CA81665-01 and RR11823-03. M.P.W. acknowledges support from genome training grant T32HG000035-05. Saccharomyces cerevisiae strain BJ5460 was a generous gift from Steve Hahn of the Fred Hutchinson Cancer Research Center (Seattle, WA).

Author information

Author notes

  1. Michael P. Washburn and Dirk Wolters: These authors contributed equally to this work.

Authors and Affiliations

  1. Syngenta Agricultural Discovery Institute, 3115 Merryfield Row, Suite 100, San Diego, 92121, CA

    Michael P. Washburn, Dirk Wolters & John R. Yates III

  2. Department of Cell Biology SR11, 10550 North Torrey Pines Road

    John R. Yates III

  3. The Scripps Research Institute, La Jolla, 92037, CA

    John R. Yates III

Authors
  1. Michael P. Washburn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dirk Wolters
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John R. Yates III
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John R. Yates III.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Washburn, M., Wolters, D. & Yates, J. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19, 242–247 (2001). https://doi.org/10.1038/85686

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/85686

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing