酪氨酸磷酸化,一种常见的蛋白质翻译后修饰过程,参与各种生物过程。然而,酪氨酸磷酸化蛋白质的丰度非常低,通过质谱(MS)进行鉴定是困难的;因此,通常需要毫克的起始材料用于它们的富集。例如,酪氨酸磷酸化在T细胞信号转导中起重要作用。然而,来自生物组织样本的原代T细胞数量很少,这些细胞难以培养和扩增;因此,T细胞信号转导的研究通常在永生化细胞系上进行,可以大大扩展。然而,来自永生化细胞系的数据不能完全模拟在真实生理状态下观察到的信号转导过程,它们通常会得出与原代T细胞完全不同的结论。因此,开发了一种高度敏感的蛋白质组学方法,用于研究原代T细胞中的酪氨酸磷酸化修饰信号。为了解决T细胞数量有限的问题,首先为隔离优化了一个全面的协议,激活,和从小鼠脾脏扩增原代T细胞。CD3+原代T细胞被成功分选;超过91%的收集的T细胞在第2天被充分激活,并且T细胞的数量在第4天扩增至超过7倍。接下来,为了解决酪氨酸磷酸化蛋白丰度低的问题,我们使用SH2-超结合剂亲和富集和固定化Ti4+亲和层析(Ti4+-IMAC)富集了与抗CD3和抗CD28共同刺激的原代T细胞的酪氨酸磷酸化多肽.使用纳米级液相色谱-串联质谱法(nanoLC-MS/MS)解析这些多肽。最后,在1mg蛋白中成功鉴定出282个酪氨酸磷酸化位点,包括T细胞受体膜蛋白CD3胞内区域的免疫受体酪氨酸活化基序(ITAM)上的许多酪氨酸磷酸化位点,以及ZAP70,LAT,VAV1和在共刺激条件下与信号转导相关的其他蛋白质。总之,为了解决初级细胞数量有限的技术问题,低丰度的酪氨酸磷酸化蛋白质,和MS检测的困难,我们开发了一种全面的蛋白质组学方法,用于深入分析原代T细胞中酪氨酸磷酸化修饰信号。该协议可以应用于映射与生理状态密切相关的信号转导网络。
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 Ti4+affinity chromatography (Ti4+-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.