ヒドロキシ酸修飾酸化金属クロマトグラフィーと脱リン酸化反応を用いたリン酸化プロテオミクス

抄録

Recent advances in MS-based proteomics coupled with phosphopeptide enrichment methods allow the simultaneous identification of hundreds of phosphorylation sites. Previously, we reported a novel phosphopeptide enrichment method using aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) ^{1), 2)}. This approach drastically reduces the binding of non-phosphorylated peptides to metal oxide and enriches phosphopeptides directly from crude biological samples such as cell lysates. In addition, we optimized elution conditions for phosphopeptides captured by HAMMOC to enlarge the phosphoproteome coverage. The optimized HAMMOC elution conditions provided a 1.6-fold increase in phosphopeptide number and a 1.9-fold increase in total peak area of phosphopeptides in comparison with the results obtained under the conventional conditions using ammonium hydroxide and phosphate buffers. By those improvements of phosphopeptide enrichment methods, over 3,000 non-redundant phosphopeptides could be identified from 100ug proteins from HeLa cell lysates at present. We also focused on the identification of multiply phosphorylated peptides, which are generally difficult to be identified by MS2 mode because the labile phosphate groups lead to the lack of b- and y-ion series. Neutral loss-triggered MS3 and multistage activation approaches have been used to generate more b- and y-ions. However, in our hand, these approaches did not show significant improvement because of the less number of the precursor ions selected for the fragmentation. On the other hand, alkaline treatments prior to LC-MS analysis, which generates dehydroalanine and dehydrobutyrine from pSer and pThr, respectively, improved to identify multiply phosphorylated peptides without using the multistage activation mode. This approach was also applied to HAMMOC to elute phosphopeptides. 1) N. Sugiyama et al., Mol. Cell. Proteomics, 6, 1103-1109(2007) 2) J. Rappsilber et al., Nat Protoc., 2, 1890-1906(2007)