若手研究者セッション
第1日 6月10日（月） 15:15～15:30 B会場（中ホール200）
- 1B-O1-1515（1P-31）
キナーゼリン酸化部位周辺の局所構造プロファイリングのための限定的脱リン酸化法の開発
(1京大院薬・ 2医薬基盤健栄研)
o平沼侑奈1・ 小形公亮1・ 石濱泰1,2
Although reversible phosphorylation is an important post-translational modification, specific roles and 3D structural characteristics of only a few phosphorylation sites have been revealed. In this study, we developed a phosphorylation site profiling method using an in vitro dephosphorylation reaction with a phosphatase and mass spectrometry to evaluate the structural features surrounding the phosphorylation site. We applied this method to profile recombinant Bruton’s tyrosine kinase (BTK). First, BTK was treated with lambda phosphatase for 0, 15, 30, and 60 minutes to perform a dephosphorylation reaction. Thereafter, BTK was digested and desalted, and the resulting peptides were measured by LC/MS/MS. From the obtained peak areas of phosphorylated peptides, the dephosphorylation ratio of phosphorylation sites on BTK was calculated for each treatment time, and a time-dependent dephosphorylation reaction profile was obtained. However, under these experimental conditions, the dephosphorylation reaction tends to saturate, making it difficult to observe a large difference in the dephosphorylation efficiency. Therefore, we optimized the amount of lambda phosphatase added and the dephosphorylation reaction time. In addition to these results, we will also report the results of profiling the in vitro dephosphorylation efficiency of purified proteins denatured under various conditions.