The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function1. To date, generation of large-scale protein–protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression2,3,4. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale5,6. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies3,4. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.
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Pawson, T. & Nash, P. Protein–protein interactions define specificity in signal transduction. Genes Dev. 14, 1027–1047 (2000).
Fields, S. & Song, O. A novel genetic system to detect protein–protein interactions. Nature 340, 245–246 (1989).
Uetz, P. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).
Ito, T. et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl Acad. Sci. USA 98, 4569–4574 (2001).
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).
Mann, M., Hendrickson, R. C. & Pandey, A. Analysis of proteins and proteomes by mass spectrometry. Annu. Rev. Biochem. 10, 437–473 (2001).
Gustin, M. C., Albertyn, J., Alexander, M. & Davenport, K. MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62, 1264–1300 (1998).
Morgan, D. O. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell. Dev. Biol. 13, 261–291 (1997).
McMillan, J. N. et al. The morphogenesis checkpoint in Saccharomyces cerevisiae: cell cycle control of Swe1p degradation by Hsl1p and Hsl7p. Mol. Cell. Biol. 19, 6929–6939 (1999).
Philips, J. & Herskowitz, I. Identification of Kel1p, a kelch domain-containing protein involved in cell fusion and morphology in Saccharomyces cerevisiae. J. Cell. Biol. 143, 375–389 (1998).
Jorgensen, P. & Tyers, M. The fork’ed path to mitosis. Genome Biol. 1 1022.1–1022.4 (2000).
Alexandru, G., Uhlmann, F., Mechtler, K., Poupart, M. & Nasmyth, K. Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast. Cell 105, 459–472 (2001).
Zhou, B. B. & Elledge, S. J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439 (2000).
Prakash, S. & Prakash, L. Nucleotide excision repair in yeast. Mutat. Res. 451, 13–24 (2000).
Thelen, M. P., Venclovas, C. & Fidelis, K. A sliding clamp model for the Rad1 family of cell cycle checkpoint proteins. Cell 96, 769–770 (1999).
Koegl, M. et al. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644 (1999).
Ortolan, T. G. et al. The DNA repair protein Rad23 is a negative regulator of multi-ubiquitin chain assembly. Nature Cell Biol. 2, 601–608 (2000).
Tyers, M. & Rottapel, R. VHL: a very hip ligase. Proc. Natl Acad. Sci. USA 96, 12230–12232 (1999).
Emili, A., Schieltz, D. M., Yates, J. R. & Hartwell, L. H. Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asf1. Mol. Cell 7, 13–20 (2001).
Hu, F., Alcasabas, A. A. & Elledge, S. J. Asf1 links Rad53 to control of chromatin assembly. Genes Dev. 15, 1061–1066 (2001).
Marsolier, M. C., Roussel, P., Leroy, C. & Mann, C. Involvement of the PP2C-like phosphatase Ptc2p in the DNA checkpoint pathways of Saccharomyces cerevisiae. Genetics 154, 1523–1532 (2000).
Durocher, D., Henckel, J., Fersht, A. R. & Jackson, S. P. The FHA domain is a modular phosphopeptide recognition motif. Mol. Cell 4, 387–394 (1999).
Zhao, X., Chabes, A., Domkin, V., Thelander, L. & Rothstein, R. The ribonucleotide reductase inhibitor Sml1 is a new target of the Mec1/Rad53 kinase cascade during growth and in response to DNA damage. EMBO J. 20, 3544–3553 (2001).
Beaudenon, S. L., Huacani, M. R., Wang, G., McDonnell, D. P. & Huibregtse, J. M. Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 6972–6979 (1999).
Bader, G. et al. BIND—The biomolecular interaction network database. Nucleic Acids Res. 29, 242–245 (2001).
Mewes, H. W. et al. MIPS: a database for genomes and protein sequences. Nucleic Acids Res. 28, 37–40 (2000).
Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. The large-scale organization of metabolic networks. Nature 407, 651–654 (2000).
Chervitz, S. A. et al. Comparison of the complete protein sets of worm and yeast: orthology and divergence. Science 282, 2022–2028 (1998).
Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 293, 2101–2105 (2001).
Wilm, M. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996).
We thank J. Chen, B. Kuehl, H. Li, V. Lay, B. Tuekam, S. Zhang, M. Patel, P. O'Donnell, I. Dutschek, U. Friedrich, M. Hansen, J. Brønd, H. Lieu, R. Woolstencroft, L. Harrington, F. Sicheri, A. Breitkreutz, C. Boone, B. Andrews and T. Hughes for discussions and/or technical assistance. This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR), the Ontario Research and Development Challenge Fund and MDS-Sciex to T.P., D.D., C.H. and M.T. T.P. is a Distinguished Scientist of the CIHR; M.F.M. is a CIHR Scientist; D.D. is a Canada Research Chair in Proteomics, Bioinformatics and Functional Genomics and a Hitchings-Elion fellow of the Burroughs-Wellcome Fund; and M.T. is a Canada Research Chair in Biochemistry.
Employment, personal financial interests and funding.
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Ho, Y., Gruhler, A., Heilbut, A. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002) doi:10.1038/415180a
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