Wilson-Grady Joshua T, Villén Judit, Gygi Steven P
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Proteome Res. 2008 Mar;7(3):1088-97. doi: 10.1021/pr7006335. Epub 2008 Feb 8.
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.
磷酸化是真核细胞中许多事件的关键调节因子。从模式生物中获取大规模磷酸化数据集可以确定保守的调节输入并揭示激酶-底物关系。在这里，我们首次对裂殖酵母粟酒裂殖酵母进行了大规模磷酸化分析。用噻苯达唑处理过的细胞中的蛋白质通过制备型SDS-PAGE分离，并用胰蛋白酶消化。所得肽段采用IMAC或TiO2磷酸肽富集方法处理，然后使用LTQ-Orbitrap质谱仪通过LC-MS/MS进行分析。总共从1194种蛋白质中鉴定出2887个不同的磷酸化位点，在肽段水平估计的错误发现率<0.5%。本文对两种不同的富集方法进行了比较，支持了它们具有互补性这一发现。最后，对磷酸化位点进行了磷酸化特异性基序和进化保守性检查。这些分析表明，粟酒裂殖酵母中鉴定出的基序和特定磷酸化事件都是保守的，并预测了哺乳动物中的新磷酸化。
