应用于原代T细胞酪氨酸磷酸化蛋白质组的高灵敏度分析方法

A highly sensitive approach for the analysis of tyrosine phosphoproteome in primary T cells

酪氨酸磷酸化在T细胞的信号转导过程中发挥着重要的作用,然而其丰度较低,鉴定困难。生物组织样本中分离获得的原代T细胞数量较少,培养和扩增的难度较大,因而常使用永生化细胞系来研究T细胞的酪氨酸磷酸化介导的信号转导过程,这通常会导致获得与原代T细胞相差较大的结论。因此,本研究发展了一种解析原代T细胞酪氨酸磷酸化修饰信号的高灵敏度的蛋白质组学方法。首先,针对原代T细胞数量有限的问题,本研究优化了一套从小鼠脾脏中分离、活化和扩增T细胞的完整流程,第4天时T细胞数量扩增到7倍以上;其次,针对酪氨酸磷酸化修饰丰度较低、不易被质谱检测的难题,本研究利用SH2超亲体(SH2-superbinder)亲和富集和固定化钛离子亲和色谱(Ti^(4+)-IMAC)技术对抗CD3和抗CD28单克隆抗体共刺激条件下的原代T细胞多肽样品进行了酪氨酸磷酸化多肽富集,并结合纳升液相色谱-串联质谱(nanoLC-MS/MS)进行解析。最终成功从1 mg蛋白质中鉴定到282个酪氨酸磷酸化位点,其中包含T细胞受体膜蛋白CD3胞内区的免疫受体酪氨酸激活序列(ITAM)上的多个酪氨酸磷酸化位点,以及信号转导相关蛋白ZAP70、LAT、VAV1等的重要位点信息。综上,本研究发展了一套深度解析原代T细胞中的酪氨酸磷酸化修饰的高灵敏度蛋白质组学分析流程,有望应用于绘制更接近生理状态下的信号转导网络。

Tyrosine phosphorylation,a common post-translational modification process for proteins,is involved in a variety of biological processes.However,the abundance of tyrosine-phosphorylated proteins is very low,making their identification by mass spectrometry(MS)is difficult;thus,milligrams of the starting material are often required for their enrichment.For example,tyrosine phosphorylation plays an important role in T cell signal transduction.However,the number of primary T cells derived from biological tissue samples is very small,and these cells are difficult to culture and expand;thus,the study of T cell signal transduction is usually carried out on immortalized cell lines,which can be greatly expanded.However,the data from immortalized cell lines cannot fully mimic the signal transduction processes observed in the real physiological state,and they usually lead to conclusions that are quite different from those of primary T cells.Therefore,a highly sensitive proteomic method was developed for studying tyrosine phosphorylation modification signals in primary T cells.To address the issue of the limited T cells numbers,a comprehensive protocol was first optimized for the isolation,activation,and expansion of primary T cells from mouse spleen.CD3+primary T cells were successfully sorted;more than 91%of the T cells collected were well activated on day 2,and the number of T cells expanded to over 7-fold on day 4.Next,to address the low abundance of tyrosine-phosphorylated proteins,we used SH2-superbinder affinity enrichment and immobilized Ti 4+affinity chromatography(Ti^(4+)-IMAC)to enrich the tyrosine-phosphorylated polypeptides of primary T cells that were co-stimulated with anti-CD3 and anti-CD28.These polypeptides were resolved using nanoscale liquid chromatography-tandem mass spectrometry(nanoLC-MS/MS).Finally,282 tyrosine phosphorylation sites were successfully identified in 1 mg of protein,including many tyrosine phosphorylation sites on the immunoreceptor tyrosine-based activation motif(ITAM)in the intracellular region of the T cell receptor membrane protein CD3,as well as the phosphotyrosine sites of ZAP70,LAT,VAV1,and other proteins related to signal transduction under costimulatory conditions.In summary,to solve the technical problems of the limited number of primary cells,low abundance of tyrosine-phosphorylated proteins,and difficulty of detection by MS,we developed a comprehensive proteomic method for the in-depth analysis of tyrosine phosphorylation modification signals in primary T cells.This protocol may be applied to map signal transduction networks that are closely related to physiological states.