Phosphoproteome analysis of fission yeast.
Phosphorylation is a key regulator of many events in eukaryotic cells. The acquisition of large-scale phosphorylation data sets from model organisms can pinpoint conserved regulatory inputs and reveal kinase-substrate relationships. Here, we provide the first large-scale phosphorylation analysis of the fission yeast, Schizosaccharomyces pombe. Protein from thiabendazole-treated cells was separated by preparative SDS-PAGE and digested with trypsin. The resulting peptides were subjected to either IMAC or TiO2 phosphopeptide enrichment methods and then analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer. In total, 2887 distinct phosphorylation sites were identified from 1194 proteins with an estimated false-discovery rate of <0.5% at the peptide level. A comparison of the two different enrichment methods is presented, supporting the finding that they are complementary. Finally, phosphorylation sites were examined for phosphorylation-specific motifs and evolutionary conservation. These analyses revealed both motifs and specific phosphorylation events identified in S. pombe were conserved and predicted novel phosphorylation in mammals.
Wilson-Grady
Joshua T
JT
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Villén
Judit
J
Gygi
Steven P
SP
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D020543:Proteome; D012568:Schizosaccharomyces / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D005656:Fungal Proteins; D010750:Phosphoproteins; D020543:Proteome