Citation: SUN Jie, ZHANG Wan-Jun, SHI Zhao-Mei, QIN Wei-Jie, QIAN Xiao-Hong. A Novel Method for Analysis of Tyrosine Phosphopeptides Based on a Centrifugal Enrichment Device[J]. Chinese Journal of Analytical Chemistry, ;2017, 45(10): 1434-1440. doi: 10.11895/j.issn.0253-3820.170139 shu

A Novel Method for Analysis of Tyrosine Phosphopeptides Based on a Centrifugal Enrichment Device

Beijing Institute of Radiation Medicine, National Center for Protein Sciences(Beijing), State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing 102206, China

Received Date: 10 March 2017
Revised Date: 16 May 2017

Fund Project: This work was supported by the National Key Program for Basic Research of China (Nos.2013CB911204, 2016YFA0501403) and the National Natural Science Foundation of China (Nos.21235001, 21405175, 21675172).

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.
- Phosphotyrosine,
- Enrichment,
- Mass spectrometry,
- Proteome,
- Post-translation modification
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