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

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 key regulatory roles in numerous signal transduction processes and in occurrence & development of tumor. The study of tyrosine phosphorylation and the 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 novel centrifugal device was developed by combination of application of titanium dioxide (TiO 2 ) and C 18 reverse phase packing materials for phosphopeptides enrichment and separation, which led to simplified procedure, reduced sample loss and minimized interference. This centrifugal device was made of pipette tips, adapters and eppendorf tubes (1.5 mL). 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, a total of 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.