Application of chemical probes for the analysis of protein tyrosine phosphatase

• Main Authors: Long-Yuan Chen, 陳龍源
• Other Authors: Jing-Jer Lin
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/54079470518388962892

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 104 === Protein tyrosine phosphorylation is a common post-translational modification of proteins. It is a dynamic, reversible process which is regulated by protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTPs). PTP is an important enzyme family that regulates tyrosine phosphorylation status in cells. Although the protein levels of PTPs can be determined by mass spectrometry, the analysis cannot precisely profile the functional state of PTPs in living cells. To overcome the hurdle, activity-based protein profiling (ABPP) was introduced as a tool to profile PTP activities. Here, we have synthesized a series of maleimide-related probes using maleimide, maleate, and, fumarate as the recognition moiety. We evaluate the labeling efficiency and specificity of these probes using purified PTPs. We found the maleate-based chemical probes reacted with the catalytic residue of tyrosine phosphatse and labeled different tyrosine phosphatases. However, since these probes also labeled other non-PTPs, the specificities of these probes have to be further improved. In addition, we have also used a 2-fluoromethyl phosphotyrosine (2-FMPT), which was developed previously in our lab, to establish an activity measuring system for dual-specific phosphatase (DUSP). Using DUSP14 as the model, we found the activity of DUSP14 can be detected directly in cell lysates through combination of activity probe-labeling and immunoprecipitation using a specific antibody against DUSP14.