News and Publications

Probing Cellular Function With Proteomics

S. Anderson, C. Delahunty, L. Florens, H. Liu, D. Lin, M.J. MacCoss, W.H. McDonald, Z.Y. Park, R. Sadygov, A. Saraf, D.L. Tabb, J. Venable, C. Wu, W.H. Zhu, J.R. Yates III

Genomic and expressed sequence tagging projects are providing a sequence infrastructure that is changing how protein biochemistry is practiced. Data produced in these projects and new developments in mass spectrometry provide the cornerstones that fuel the proteomics revolution.

For proteomic-scale work, proteins are usually identified by using 1 of 2 methods: peptide mass mapping or peptide tandem mass spectrometry. In both methods, the protein sample is digested with endoproteases to produce smaller and more easily analyzed peptides. In peptide mass mapping, the sequence specificity of the proteases is used to compare the sizes of peptides in the sample with the sizes of predicted peptides from proteins within the database. Although this technique allows rapid identification of peptides, it requires a relatively pure sample, and because it depends on measurements of molecular weight, it requires multiple peptides for identification. In contrast, peptide tandem mass spectrometry involves fragmentation of individual peptides, and identification of a peptide is based on the amino acid sequence of that specific peptide. Because every peptide can "stand alone" for an identification, much more complex mixtures of proteins can be analyzed.

By coupling these abilities of the tandem mass spectrometer to multidimensional chromatographic separation, we can analyze extremely complex mixtures of proteins. We termed this integrated system multidimensional protein identification technology or MudPIT. We routinely use MudPIT to identify the protein components of a wide variety of samples. The samples can vary in complexity from relatively simple purified protein complexes all the way to whole-cell lysates, and we address a number of important biological questions.

Two of our projects involve understanding the life cycles of the microorganisms that cause malaria and anthrax and the interactions of these pathogens with the host. Other collaborative efforts include proteomic analyses of extremophile bacteria and literally hundreds of purified protein complexes from both the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. Two other human diseases that we are collaboratively studying are cystic fibrosis and cataract formation in the lens.

Although the technology allows routine identification of proteins, much of the regulation of protein activity takes place through modifications that occur after translation. Because these modifications cannot, in general, be predicted on the basis of the protein or nucleotide sequence, we expanded our technology to allow their identification. This new process is simple, powerful, and general and can be applied to complicated protein mixtures. A protein or protein mixture is proteolytically digested by using 3 different enzymes: 1 that cleaves in a site-specific manner and 2 that cleave nonspecifically. By using MudPIT analysis of this extremely complex mixture of peptides, we can collect data for the maximum number of peptides possible, including the modified peptides. The method has the added benefit of often producing multiple overlapping peptides pointing to the same site of modification. Unlike other reported strategies for the analysis of posttranslational modifications, this strategy is not limited to searching for a particular type of modification.

For demonstration, we applied this approach to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated by using an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites with known stoichiometry as low as 10% were detected. Eighteen sites of 4 different types of modification were detected on 3 of the 5 proteins in the simple protein mixture, 3 of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded 8 sites containing 3 different types of modifications. In the lens tissue, 270 proteins were identified, and 12 different proteins contained a total of 74 sites of modification. Modifications identified in the lens proteins included serine, threonine, and tyrosine phosphorylation; arginine and lysine methylation; lysine acetylation; and methionine, tyrosine, and tryptophan oxidations.

The computational resources required to handle these volumes of data are significant. Another focus of our group is algorithmic enhancements to increase speed and flexibility and to better match modified protein sequences. Organizing, summarizing, and presenting data from these experiments become even greater challenges as the scope of the experiments expand.

PUBLICATIONS

Boddy, M.N., Gaillard, P.H.L., McDonald, W.H., Shanahan, P., Yates, J.R. III, Russell, P. Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell 107:537, 2001.

Cheeseman, I.M., Brew, C., Wolyniak, M., Desai, A., Anderson, S., Muster, N., Yates, J.R., Huffaker, T.C., Drubin, D.G., Barnes, G. Implication of a novel multiprotein Dam1p complex in outer kinetochore function. J. Cell Biol. 155:1137, 2001.

Cheeseman, I.M., Enquist-Newman, M., Muller-Reichert, T., Drubin, D.G., Barnes, G. Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex. J. Cell Biol. 152:197, 2001.

Chen, W., Laidig, K.E., Park, Y., Park, K., Yates, J.R. III, Lamont, R.J., Hackett, M. Searching the Porphyromonas gingivalis genome with peptide fragmentation mass spectra. Analyst 126:52, 2001.

Dongre, A.R., Kovats, S., deRoos, P., McCormack, A.L., Nakagawa, T., Paharkova-Vatchkova, V., Eng, J., Caldwell, H., Yates, J.R. III, Rudensky, A.Y. In vivo MHC class II presentation of cytosolic proteins revealed by rapid automated tandem mass spectrometry and functional analyses. Eur. J. Immunol. 31:1485, 2001.

Emili, A., Schieltz, D.M., Yates, J.R. III, Hartwell, L.H. Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asf1. Mol. Cell 7:13, 2001.

Friedman, D.B., Kern, J.W., Honeycut, B.J., Vinh, D.B., Crawford, D.K., Steiner, E., Schieltz, D., Yates, J.R. III, Resing, K.A., Ahn, N.G., Winey, M., Davis, T.N. Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain. J. Biol. Chem. 276:17958, 2001.

Giometti, C.S., Reich, C., Tollaksen, S., Babnigg, G., Lim, H., Yates, J.R. III, Olsen, G. Structural modifications of Methanococcus jannaschii flagellin proteins revealed by proteome analysis. Proteomics 1:1033 2001.

Honey, S., Schneider, B.L., Schieltz, D.M., Yates, J.R., Futcher, B. A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex. Nucleic Acid Res. 29:E24, 2001.

Lim, H., Yates, J.R. III. Mass spectrometry: analysis of two-dimensional protein gels. Encyclopedia of Life Sciences. 2001. Available at www.els.net.

Lin, D., Alpert, A., Yates, J.R. III. Multidimensional protein indentification technology as an effective tool for proteomics. Am. Genomic/Proteomics Technol. 1:38, 2001.

Liu, H., Lin, D., Yates, J.R. III. Multidimensional separations for protein/peptide analysis in the post-genomic era. Biotechniques 32:898, 2002.

MacCoss, M.J., McDonald, W.H., Saraf, A., Sadygov, R., Clark, J.M., Tasto, J.J., Gould, K.L., Wolters, D., Washburn, M., Weiss, A., Clark, J.I., Yates, J.R. III. Shotgun Identification of protein modifications from protein complexes and lens tissue. Proc. Natl. Acad. Sci. U. S. A. 99:7900, 2002.

MacCoss, M.J., Yates, J.R. III. Proteomics: analytical tools and techniques. Curr. Opin. Clin. Nutr. Metab. Care 4:369, 2001.

McDonald, W.H., Ohi, R., Miyamoto, D.T., Mitchison, T.J., Yates, J.R. III. Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. Int. J. Mass Spectrom. 219:245, 2002.

Osada, S., Sutton, A., Muster, N., Brown, C.E., Yates, J.R. III, Sternglanz, R., Workman, J.L. The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1. Genes Dev. 15:3155, 2001.

Saitoh, S., Chabes, A., McDonald, W.H., Thelander, L., Yates, J.R., Russell, P. Cid13 is a cytoplasmic poly(A) polymerase that regulates ribonucleotide reductase mRNA. Cell 109:563, 2002.

Segarini, P.R., Nesbitt, J.E., Li, D., Hayes, L.G., Yates, J.R. III, Carmichael, D.E. The low density lipoprotein receptor-related protein/a₂-macroglobulin receptor is a receptor for connective tissue growth factor. J. Biol. Chem. 276:40659, 2001.

Tabb, D.L., McDonald, W.H., Yates, J.R. III. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 1:21, 2002.

Verma, R., McDonald, H., Yates, J.R. III, Deshaies, R.J. Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields active S phase cyclin-Cdk. Mol. Cell 8:439, 2001.

Wolters, D., Washburn, M.P., Yates, J.R. III. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73:5683, 2001.