基于离心式富集装置的酪氨酸磷酸肽分析新方法

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

酪氨酸磷酸化及其相应激酶活性的研究在抗肿瘤药物靶点的研发中具有重要意义。由于酪氨酸磷酸化仅占蛋白质总磷酸化含量的不足0.1%,因此规模化的酪氨酸磷酸化鉴定面临着重大技术挑战。本研究构建了TiO_2串联C_(18)反相填料的离心式富集装置,结合抗体免疫沉淀法,建立了酪氨酸磷酸肽的富集策略。此新型富集装置由吸头、适配器和离心(EP)管组成,将TiO_2富集磷酸肽和C_(18)填料反相分离磷酸肽有机结合,以离心的方式进行样品的上样、清洗、洗脱和分离,再通过抗酪氨酸磷酸化抗体进一步特异性富集酪氨酸磷酸肽,从而实现了酪氨酸磷酸肽的高效富集和大规模质谱鉴定。通过离心式富集装置简化了实验步骤,减少了样品损失和人为因素干扰;而且离心式、平行化的样品处理方式可显著提高分析通量。将此策略成功用于小鼠肝脏蛋白质酪氨酸磷酸化肽段的富集和质谱鉴定,在5 mg鼠肝蛋白中共鉴定出967个酪氨酸磷酸化位点,对应545个蛋白质,显示了其在蛋白质组学研究中的应用潜力。

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（TiO_2） 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.