色谱2024，Vol.42Issue(7)：693-701,9.DOI:10.3724/SP.J.1123.2024.01016
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
摘要
Abstract
Tyrosine phosphorylation,a common post-translational modification process for pro-teins,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.Howev-er,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 suc-cessfully 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 immobi-lized Ti4+affinity chromatography(Ti4+-IMAC)to enrich the tyrosine-phosphorylated polypep-tides of primary T cells that were co-stimulated with anti-CD3 and anti-CD28.These polypep-tides 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(IT AM)in the intracellular region of the T cell receptor membrane pro-tein CD3,as well as the phosphotyrosine sites of ZAP70,LAT,VAV1,and other proteins relat-ed 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.关键词
纳升液相色谱-串联质谱/酪氨酸磷酸化/原代T细胞/共刺激/信号转导Key words
nanoscale liquid chromatography-tandem mass spectrometry(nanoLC-MS/MS)/tyrosine phosphorylation/primary T cell/co-stimulation/signal transduction分类
化学化工引用本文复制引用
梁富超,柯弥,田瑞军..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[J].色谱,2024,42(7):693-701,9.基金项目
国家重点研发计划(2021YFA1301601,2021YFA1301602,2021YFA1302603) （2021YFA1301601,2021YFA1301602,2021YFA1302603）
国家自然科学基金(92253304,22125403) （92253304,22125403）
深圳市科技创新委员会(JSGGZD20220822095200001,JCYJ20210324120210029).National Key Research and Development Program of China(Nos.2021YFA1301601,2021YFA1301602,2021YFA1302603) （JSGGZD20220822095200001,JCYJ20210324120210029）
National Natural Science Foundation of China(Nos.92253304,22125403) （Nos.92253304,22125403）
Shenzhen Innovation of Science and Technology Commission(Nos.JSGGZD20220822095200001,JCYJ20210324120210029). （Nos.JSGGZD20220822095200001,JCYJ20210324120210029）
