A Novel Method for Analysis of Tyrosine Phosphopeptides Based on a Centrifugal Enrichment Device
10.11895/j.issn.0253-3820.170139
- VernacularTitle:基于离心式富集装置的酪氨酸磷酸肽分析新方法
- Author:
Jie SUN
1
;
Jun Wan ZHANG
;
Mei Zhao SHI
;
Jie Wei QIN
;
Hong Xiao QIAN
Author Information
1. 军事医学科学院放射与辐射医学研究所
- Keywords:
Phosphotyrosine;
Enrichment;
Mass spectrometry;
Proteome;
Post-translation modification
- From:
Chinese Journal of Analytical Chemistry
2017;45(10):1434-1440
- CountryChina
- Language:Chinese
-
Abstract:
Protein tyrosine phosphorylation is an important post-translational modification and has become one of the most active areas in proteome research in recent years. Protein tyrosine phosphorylation plays a key regulatory role in the numerous signal transduction processes and in the occurrence & development of tumor. The study of tyrosine phosphorylation and activity of it corresponding tyrosine kinases is of great significance for the research of drug targets for cancer treatment. However, tyrosine phosphorylation only represents less than 0. 1% of the total cellular protein phosphorylation. Therefore, there is a great challenge to identify tyrosine phosphorylation in real complex samples. In this work, a new centrifugal device by combining application of titanium dioxide ( TiO2 ) and C18 reversed phase packing materials for phosphopeptide enrichment and separation was developed, which led to simplified procedure, reduced sample loss and minimized interference.This new centrifugal device was made of pipette tips, adapters and Eppendorf tubes. Sample loading, phosphopeptides enrichment, washing, eluting and separation were combined into this device and could be achieved by simple centrifugation. This device was capable of paralleled sample processing with improved analysis throughput. Tandem enrichment by anti-phosphotyrosine antibody resulted in efficient enrichment and large scale identification of phosphotyrosine peptides by mass spectrometry. As a result, 967 phosphotyrosine sites corresponding to 545 proteins were successfully identified from 5 mg of mouse liver proteins, demonstrating the robustness and potential of this new strategy.
