科研之友
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摘要
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 C-18 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.
- 出版日期2017-10
- 单位北京中医药大学
