News and Publications

Probing Cellular Function With Proteomics

J. Yates, M. MacCoss, N. Muster, A. Saraf, C. Deluhunty, S. Anderson, H. McDonald, D. Lin, H. Liu, R. Sadygov, L. Florens, E. Durr

Genomic and expressed sequence tagging projects are providing a sequence infrastructure that is changing how protein biochemistry is practiced. Separation techniques and mass spectrometers, especially tandem mass spectrometers, are the other driving factors in this change. Data produced by tandem mass spectrometers provide independent pieces of information on the components of a mixture. For protein mixtures, the identities of the components can be determined by using appropriate software to correlate tandem mass spectra to peptide sequences in a database. In this approach, enzymatic digestion of a protein mixture is followed by separation of the resulting mixture of peptides and tandem mass spectrometry. Data-dependent operation of the spectrometer allows the highly efficient acquisition of tandem mass spectra. This approach has been successfully applied to large protein complexes and proteins from a total cell lysate, that is, the proteome.

We developed an unbiased method for rapid and comprehensive proteome analysis via multidimensional liquid chromatography, tandem mass spectrometry, and database searching via the SEQUEST algorithm. This integrated approach is known as multidimensional protein identification technology or MudPIT. MudPIT was applied to the proteome of Saccharomyces cerevisiae strain BJ5460 grown to mid log phase and yielded the most comprehensive proteome analysis to date. A total of 1484 unique proteins were detected and identified. Categorization of these proteins indicated that this technology can be used to detect and identify proteins rarely seen in proteome analysis, including integral membrane proteins from several cellular compartments and proteins present in low concentrations, such as transcription factors and protein kinases.

Of particular interest was our identification of 131 proteins with 3 or more predicted transmem-brane domains. The resulting data allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT not only is ideal for proteome analysis but also can be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of molecules. We are applying this method to many important biological problems, including host-pathogen interactions in malaria and the effects of aging in human lenses.

PUBLICATIONS

Bark, S.J., Muster, N., Yates, J.R. III, Siuzdak, G. High-temperature mass mapping using a thermophilic protease. J. Am. Chem. Soc. 123:1774, 2001.

Haynes, P., Yates, J.R. III. Proteome profiling: Pitfalls and progress. Yeast 17:81, 2000.

Hefner, Y., Borsch-Haubold, A.G., Murakami, M., Wilde, J.I., Pasquet, S., Schieltz, D., Ghomashchi, F., Yates, J.R. III, Armstrong, C.G., Paterson, A., Cohen, P., Fukunaga, R., Hunter, T., Kudo, I., Watson, S.P., Gelb, M.H. Serine 727 phosphorylation and activation of cytosolic phospholipase A₂ by MNK1-related protein kinases. J. Biol. Chem. 275:37542, 2000.

Krahmer, M.T., Walters, J.J., Fox, K.F., Fox, A., Creek, K.E., Pirisi, L., Wunschel, D.S., Smith, R.D., Tabb, D.L., Yates, J.R. III. MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products. Anal. Chem. 72:4033, 2000.

McDonald, W.H., Yates, J.R. III. Proteomic tools for cell biology. Traffic 1:747, 2000.

Tabb, L., Eng, J.K., Yates, J.R. Protein identification by SEQUEST. In: Proteome Research: Mass Spectrometry. James, P. (Ed.). Springer-Verlag, New York, 2000, p. 125.

Taylor, R.S., Wu, C.C., Hays, L.G., Eng, J.E., Yates, J.R. III, Howell, K.E. Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation. Electrophoresis 21:3441, 2000.

Todorow, Z., Spang, A., Carmack, E., Yates, J., Schekman, R. Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum. Proc. Natl. Acad. Sci. U. S. A. 97:13643, 2000.

Tong, W., Yates, J.R. Sensitive and high resolution CE/MS/MS for protein identification in complex mixtures. Chromatographia 53:S90, 2001.

Verma, R., Chen, S., Feldman, R., Schieltz, D., Yates, J., Deshaies, R.J. Proteasomal proteomics: Identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11:3425, 2000.

Washburn, M., Yates, J.R. III. Novel methods of proteome analysis: Multidimensional chromatography and mass spectrometry. In: Proteomics: A Trends Guide. Elsevier, New York, 2000, p. 27.

Washburn, M.P., Wolters, D., Yates, J.R. III. Large-scale analysis of the yeast proteome via multidimensional protein identification technology. Nat. Biotechnol. 19:242, 2001.

Washburn, M.P., Yates, J.R. III. Analysis of the microbial proteome. Curr. Opin. Microbiol. 3:292, 2000.

Whitten, M.E., Yokoyama, K., Schieltz, D., Ghomashchi, F., Lam, D., Yates, J.R. III, Palczewski, K., Gelb, M.H. Structural analysis of protein prenyl groups and associated C-terminal modifications. Methods Enzymol. 316:436, 2000.

Wu, C.C., Howell, K.E., Neville, M.C., Yates, J.R. III, McManaman, J.L. Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells. Electrophoresis 21:3470, 2000.

Wu, C.C., Yates, J.R. III, Neville, M.C., Howell, K.E. Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells. Traffic 1:769, 2000.

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