Article | Published:

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

Nature Biotechnology volume 19, pages 242247 (2001) | Download Citation

Subjects

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 optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Genomics, gene expression and DNA arrays. Nature 405, 827–836 (2000).

  2. 2.

    , , , & Expression profiles of active genes in human and mouse livers. Gene 174, 151–158 (1996).

  3. 3.

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

  4. 4.

    , , , & A sampling of the yeast proteome. Mol. Cell. Biol. 19, 7357–7368 (1999).

  5. 5.

    , , & Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 19, 1720–1730 (1999).

  6. 6.

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

  7. 7.

    , & Proteomics: theoretical and experimental considerations. Biotechnol. Prog. 15, 312–318 (1999).

  8. 8.

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

  9. 9.

    & Proteomics to study genes and genomes. Nature 405, 837–846 (2000).

  10. 10.

    & Analysis of the microbial proteome. Curr. Opin. Microbiol. 3, 292–297 (2000).

  11. 11.

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

  12. 12.

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

  13. 13.

    , , & The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 21, 1104–1115 (2000).

  14. 14.

    , , , & Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography. Electrophoresis 20, 2181–2195 (1999).

  15. 15.

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

  16. 16.

    , , , & Evaluation of two-dimensional electrophoresis-based proteome analysis. Proc. Natl. Acad. Sci. USA 97, 9390–9395 (2000).

  17. 17.

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

  18. 18.

    , & Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–70 (2000).

  19. 19.

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

  20. 20.

    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).

  21. 21.

    , & 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).

  22. 22.

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

  23. 23.

    & Novel methods of proteome analysis: multidimensional chromatography and mass spectrometry. Proteomics: A Current Trends Supplement, 28–32 (2000).

  24. 24.

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

  25. 25.

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

  26. 26.

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

  27. 27.

    , , , & 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).

  28. 28.

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

  29. 29.

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

  30. 30.

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

  31. 31.

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

  32. 32.

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

  33. 33.

    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).

  34. 34.

    , & The detection and classification of membrane-spanning proteins. Biochim. Biophys. Acta 815, 468–476 (1985).

  35. 35.

    , , , & The membrane proteins encoded by yeast chromosome III genes. FEBS Lett. 325, 112–117 (1993).

  36. 36.

    , , & Biogenesis and function of the yeast plasma-membrane H(+)-ATPase. J. Exp. Biol. 203, 155–160 (2000).

  37. 37.

    , & Three-dimensional map of the plasma membrane H+-ATPase in the open conformation. Nature 392, 840–843 (1998).

  38. 38.

    , , , & Structure of the calcium pump from sarcoplasmic reticulum at 8-Å resolution. Nature 392, 835–839 (1998).

  39. 39.

    , & Structure of the P-type ATPases. Curr. Opin. Struct. Biol. 8, 510–516 (1998).

  40. 40.

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

  41. 41.

    , , & Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405, 647–655 (2000).

  42. 42.

    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).

  43. 43.

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

  44. 44.

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

  45. 45.

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

  46. 46.

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

  47. 47.

    , , , & Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591–6596 (1999).

  48. 48.

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

  49. 49.

    , , & 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).

  50. 50.

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

  51. 51.

    , , , & 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).

  52. 52.

    , , , & 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).

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

    • Michael P. Washburn
    •  & Dirk Wolters

    These authors contributed equally to this work.

Affiliations

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

    • Michael P. Washburn
    • , Dirk Wolters
    •  & John R. Yates III
  2. Department of Cell Biology SR11, 10550 North Torrey Pines Road, The Scripps Research Institute, La Jolla, CA 92037.

    • John R. Yates III

Authors

  1. Search for Michael P. Washburn in:

  2. Search for Dirk Wolters in:

  3. Search for John R. Yates in:

Corresponding author

Correspondence to John R. Yates III.

Supplementary information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/85686

Further reading