2P-03 PDF
Monitoring kinome activities using kinase-specific substrate peptides
Protein phosphorylation catalyzed by kinases is one of the most ubiquitous post-translational modifications and plays significant roles in cellular signal transduction. Monitoring kinome activities provides us an understanding of the cell signaling, and can be beneficial for the diagnosis. Although phosphoproteomics based on LC-MS enables us to identify a large number of phosphosites, these data lack the information of responsible kinases. Thus, it is difficult to measure kinome activities by current phosphoproteomics approaches. In this study, we developed a method to monitor kinome activities using kinase-specific substrate peptides.
Firstly, we selected candidates for the kinase-specific substrates based on large scale kinase-substrate relationships obtained by in vitro kinase assay combined with phosphoproteomics. We synthesized these substrate peptides and investigated their specificity by in vitro kinase assay. As a result, our designed substrate peptides were phosphorylated by a specific kinase or kinase family in vitro. Furthermore, linearity and sensitivity for these substrate peptides were evaluated in the presence of a crude cell extract.