News and Publications

Probing Cellular Function With Proteomics

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 of protein, and because it depends on measurements of molecular weight, it requires multiple peptides for identification. In contrast, peptide tandem mass spectrometry involves fragmentation of the 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 capabilities of tandem mass spectrometry to a multidimensional chromatographic separation, we can analyze extremely complex mixtures of proteins. We termed this method 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. We profiled the proteins expressed during the life cycle of Plasmodium falciparum, the parasite that causes malaria. We found that protein expression varied quite significantly in sporozoites, trophozoites, merozoites, and gametocytes. In addition, we identified clusters of genes that have stage-specific expression, suggesting coregulation at the level of transcription. We also extended our efforts to identify membrane-bound proteins that may be potential targets for vaccine development. A large number of membrane proteins were identified.

Publications

Carlton, J.M., Angiuoli, S.V., Suh, B.B., Kooij, T.W., Pertea, M., Silva, J.C., Ermolaeva, M.D., Allen, J.E., Selengut, J.D., Koo, H.L., Peterson, J.D., Pop, M., Kosack, D.S., Shumway, M.F., Bidwell, S.L., Shallom, S.J., van Aken, S.E., Riedmuller, S.B., Feldblyum, T.V., Cho, J.K., Quackenbush, J., Sedegah, M., Shoaibi, A., Cummings, L.M., Florens, L., Yates, J.R., Raine, J.D., Sinden, R.E., Harris, M.A., Cunningham, D.A., Preiser, P.R., Bergman, L.W., Vaidya, A.B., van Lin, L.H., Janse, C.J., Waters, A.P., Smith, H.O., White, O.R., Salzberg, S.L., Venter, J.C., Fraser, C.M., Hoffman, S.L., Gardner, M.J., Carucci, D.J. Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii. Nature 419:512, 2002.

Cheeseman, I.M., Anderson, S., Jwa, M., Green, E.M., Kang, J., Yates, J.R. III, Chan, C.S., Drubin, D.G., Barnes, G. Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p. Cell 111:163, 2002.

Florens, L., Washburn, M.P., Raine, J.D., Anthony, R.M., Grainger, M., Haynes, J.D., Moch, J.K., Muster, N., Sacci, J.B., Tabb, D.L., Witney, A.A., Wolters, D., Wu, Y., Gardner, M.J., Holder, A.A., Sinden, R.E., Yates, J.R., Carucci, D.A. A proteomic view of the Plasmodium falciparum life cycle. Nature 419:520, 2002.

Verma, R., Aravind, L., Oania, R., McDonald, W.H., Yates, J.R. III, Koonin, E.V., Deshaies, R.J. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298:611, 2002.