@article{2411, keywords = {Proteomics, Binding Sites, Protein Binding, Phosphoproteins, Saccharomyces cerevisiae Proteins, Phosphotransferases, Phosphorylation, Tandem Mass Spectrometry}, author = {An Chi and Curtis Huttenhower and Lewis Geer and Joshua Coon and John Syka and Dina Bai and Jeffrey Shabanowitz and Daniel Burke and Olga Troyanskaya and Donald Hunt}, title = {Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry.}, abstract = {
We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-microg (approximately 600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from <50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.
}, year = {2007}, journal = {Proc Natl Acad Sci U S A}, volume = {104}, pages = {2193-8}, month = {02/2007}, language = {eng}, }