Global proteomic & metabolic profiling of yeast cells.
Edward Marcotte, Peng Lu, John Prince, Anu Rangan, Sherwin Chan, Dean Appling, David Hoffman
Inst. for Cell & Molec Biology, University of Texas at Austin, 2500 Speedway, Austin, TX, 78712, USA
Despite progress on monitoring global mRNA expression levels, monitoring the proteome and metabolome, which should reflect proteins' in vivo activities, is an open functional genomics challenge. Here, we describe mass spectrometry-based protein expression profiling of the yeast proteome, and 1H-13C 2D-NMR for in vitro and in vivo metabolic profiling of cells. For proteomics, mass spectrometry of tryptic peptides from the yeast proteome, combined with stable isotope incorporation, allows quantitative profiling of abundant yeast proteins. For metabolomics, approx. 200 peaks in 2D-NMR spectra of yeast cells or lysates can be reproducibly observed across differing growth conditions, allowing profiling of the most abundant yeast metabolites, including amino acids, monosaccharides, nucleotides and other small molecules. Metabolic profiling of wild-type yeast and a one-carbon metabolism mutant reveals complex metabolome changes, including differences in amino acid and sugar metabolism, sequestering of methionine and S-adenosylmethionine by the mutant, feedback inhibition of homocysteine biosynthesis by cysteine, and possible variation in the global nitrogen balances. Combining the proteomic and metabolomic data with microarray-based profiling of transcription provides a quantitative description of the intracellular state of the yeast cells, essentially creating a complex, quantitative phenotype, and we present preliminary results on integrating these diverse data.
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