Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry

An Chi, Curtis Huttenhower, Lewis Y. Geer, Joshua J. Coon, John E.P. Syka, Dina L. Bai, Jeffrey Shabanowitz, Daniel J. Burke, Olga G. Troyanskaya, Donald F. Hunt

Research output: Contribution to journalArticle

468 Scopus citations

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-μg (≈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.

Original languageEnglish (US)
Pages (from-to)2193-2198
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number7
DOIs
StatePublished - Feb 13 2007

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Network analysis
  • Yeast phosphoproteome

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