演題概要

ポスター発表

第3日 5月20日(金)  ポスター会場(いちょう)

多重安定同位体標識を用いる高深度定量リン酸化プロテオミクスにおける高感度化

(京大院薬)
o小形公亮Tsai, Chia-feng若林真樹杉山直幸石濱泰

Protein phosphorylation is well known as one of the key factors to control intracellular signal transduction that regulates various cell functions, and the in-depth analysis of the phosphoproteome of interest is indispensable to understand each biological event. Recent advances in LC-MS together with phosphopeptide enrichment allow identifying thousands of phosphopeptides in single analysis. However, it is still challenging to accomplish both the deep coverage and the high throughput measurement in quantitative phosphoproteomics for a small sample amount. In this study, we proposed a new strategy for deep and high throughput quantitative phosphoproteomics, in which multiplex isobaric stable isotope tags are incorporated within SPE tips after phosphopeptide enrichment and one-shot LC-MS analysis using meter-long monolithic silica columns is employed without pre-fractionation.
At first, we compared three different workflows with each other using HeLa lysates and Tandem Mass TagTM (TMT), such as (1) in-solution TMT labeling followed by phosphopeptide enrichment, (2) phosphopeptide enrichment followed by in-solution TMT labeling and (3) phosphopeptide enrichment followed by in-tip TMT labeling using a new protocol. As a result, the workflow (3) showed the highest sensitivity as shown in Fig. 1. This strategy is useful for deep and high-throughput quantitative phosphoproteomics for limited sample amounts.