日本質量分析学会 第66回質量分析総合討論会

Program

Poster Presentations

Day 4, May 18(Fri.)  Poster

Ultra-sensitive motif-targeting approach for measurement of tyrosine phosphorylation stoichiometry in EGFR

(1Institute of Chemistry, Academia Sinica, 2Department of Chemistry, National Taiwan University, 3Graduate School of Pharmaceutical Sciences, Kyoto University)
oYen-chen Liao1,2, Pin-lian Chiang1,2, Chia-feng Tsai3, Miao-hsia Lin1, Pei-yi Lin1, Yasushi Ishihama3, Yu-ju Chen1,2

Tyrosine phosphorylation is a key upstream regulator in signaling pathway; however, the detection of tyrosine phosphorylation remains a challenge due to its extremely low abundance compared to phosphoserine and phosphothreonine in shotgun phosphoproteomics. For in-depth delineation of the phosphorylation network, measurement of the phosphorylation stoichiometry might illustrate whether the signal-induced alteration is regulated by upstream enzyme activity or by transcriptional regulation to effect protein abundance.
Using epidermal growth factor receptor (EGFR) as study model, we developed an IP-based motif-targeting quantitative approach which integrates protein immunoprecipitation (IP), metabolic labeling, dephosphorylation by phosphatase, and tyrosine kinase reaction for stoichiometry measurement. By this strategy, the number of identified phosphotyrosine sites can be increased to 14 sites in 6 mg cell lysate, compared to 2 sites from conventional phosphoproteomics approach. We applied this approach to analyze the tyrosine phosphorylation dynamics in response to geftinib (a Tyrosine Kinase Inhibitor) treatment in PC9 cells (EGFR with exon 19 deletion mutation). The temporal profile shows that Tyr1110 and Tyr1172 sites have high stoichiometry at basal level and drop to 50% at 5 minutes. These results are consistent with western blotting. Application of this approach on comparison of wild type EGFR and its mutation subtypes is on-going.