title: [Proteomic peptide library for determination of substrate motif of casein kinase 2].
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.
authors: Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors: Manyu
authors: M
authors: Zhang
authors: Chunli
authors: C
authors: Wang
authors: Yangyang
authors: Y
authors: Bian
authors: Kai
authors: K
authors: Cheng
authors: Xiaoluan
authors: X
authors: Wei
authors: Mingliang
authors: M
authors: Ye
authors: Hanfa
authors: H
authors: Zou
mesh_terms: D020816:Amino Acid Motifs; D047390:Casein Kinase II / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D019151:Peptide Library; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D019151:Peptide Library; D010748:Phosphopeptides; D020543:Proteome; D047390:Casein Kinase II