基于离心式富集装置的酪氨酸磷酸肽分析新方法

发布时间：2018-04-17 14:47

  本文选题：酪氨酸磷酸化 + 富集　； 参考：《分析化学》2017年10期

【摘要】：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。
[Abstract]:The study of tyrosine phosphorylation and its kinase activity is of great significance in the development of antitumor drug targets.Since tyrosine phosphorylation accounts for less than 0.1% of the total protein phosphorylation, the identification of tyrosine phosphorylation on a large scale is facing great technical challenges.In this study, a centrifugal enrichment device with TiO_2 series CST18) reverse phase filler was constructed, and the enrichment strategy of tyrosine phosphopeptide was established with antibody immunoprecipitation method.This new type of enrichment device consists of suction head, adapter and centrifuge EPP-tube. It organically combines TiO_2 enrichment of phosphate peptide with CST18) filler to separate phosphate peptide in reverse phase, and carries out sample preparation, cleaning, elution and separation by centrifugation.Furthermore, the tyrosine phosphopeptide was further enriched by anti-tyrosine phosphorylation antibody, so that the high concentration of tyrosine phosphopeptide and the identification of mass spectrometry were realized.The experiment procedure is simplified by centrifugal enrichment device, and the sample loss and human interference are reduced, and the analytical flux can be significantly increased by the centrifugal and parallel sample treatment.The strategy was successfully applied to the enrichment and mass spectrometry identification of the tyrosine phosphorylated peptide segment of mouse liver protein. 967 tyrosine phosphorylation sites were identified at 5 mg mouse liver protein, corresponding to 545 proteins.It shows its potential application in proteomics research.
【作者单位】： 军事医学科学院放射与辐射医学研究所国家蛋白质科学研究中心蛋白质组学国家重点实验室北京蛋白质组研究中心;
【基金】：国家重点研发计划(Nos.2013CB911204,2016YFA0501403) 国家自然科学基金(Nos.21235001,21405175,21675172)资助~~
【分类号】：O629.72;O652.6
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本文编号：1764044