Abstract

Poster Presentations

Day 1, June 10（Mon.）　　Room P1 (Multipurpose Hall)・Room P2 (Conference Room 101+102)

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.

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