Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry.
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.
Chi
An
A
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Huttenhower
Curtis
C
Geer
Lewis Y
LY
Coon
Joshua J
JJ
Syka
John E P
JE
Bai
Dina L
DL
Shabanowitz
Jeffrey
J
Burke
Daniel J
DJ
Troyanskaya
Olga G
OG
Hunt
Donald F
DF
D001665:Binding Sites; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D010770:Phosphotransferases