Abstract: Substrate motif is of great importance in kinase-substrate recognition and contributes a lot to the understanding of signal transduction. In this study, a proteomic peptide library was generated to determine substrate motif of casein kinase 2. Firstly, whole cell lysate was digested by trypsin to generate a large number of candidate peptides, which were then incubated with alkaline phosphotase to dephosphorylate intrinsic phosphopeptides. Then, the unphosphorylated peptide mixture was incubated with casein kinase 2 (CK2) and adenosine-triphosphate (ATP) for 30 min, and the resulting phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) followed by reversed-phase liquid chromatographic separation coupled with tandem mass spectrometry detection. Finally, the 472 unique phosphopeptides and 451 unique phosphorylation sites were identified, resulting in the determination of a motif of S/T-D/E-x-D/E for CK2. This method enables accurate determination of substrate motif in a short time and can be readily applied to other kinases.

Key words: kinase, motif, peptide library, proteomics, reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), immobilized metal affinity chromatography (IMAC)