对JurkatT细胞质膜蛋白的初步研究.doc

Jurkat T细胞质膜蛋白组学研究初探 甘成军，谭江琳，胡婕，王晓娟，张翠华，张晓容，罗高兴，贺伟峰，吴军 （第三军医大学西南医院烧伤研究所，烧伤、创伤、复合伤国家重点实验室，疾病蛋白质组学重庆市市级重点实验室 重庆 400038） 摘 要: 目的 应用差异密度梯度离心方法结合一维凝胶电泳-质谱法全谱分析Jurkat T细胞质膜蛋白质组成，并对这些质膜蛋白的生理学过程和功能进行初步分析，为进一步研究Jurkat T细胞膜蛋白功能奠定基础。　 方法　首先采用差异密度梯度离心法提取Jurkat T细胞膜蛋白，然后将提取出的膜蛋白根据分子量大小通过SDS进行初步分离，再进一步将分离出的蛋白条带切下进行胶内酶解，酶解后的肽段通过液相-芯片-离子阱质谱技术进行鉴定和生物信息学分析，建立Jurkat T细胞质膜蛋白全谱图，并进一步通过GO（Gene Ontology）对这些质膜蛋白进行功能分析。 结果　成功提取了Jurkat T细胞的膜总蛋白，并建立了Jurkat T细胞质膜蛋白全谱图，共鉴定出618个质膜蛋白，经GO注释分析，其中与结合功能相关的质膜蛋白有493个，与信号转导活性相关的有186个，具有酶催化活性的有166个，具有转运活性的有137个，有些还具有酶调节活性、结构分子活性或者运动活性等，功能尚不清楚的有49个。 结论　通过差异密度梯度离心，结合一维SDS和HPLC-CHIP-MS/MS，成功建立了Jurkat T细胞质膜蛋白全谱图，并通过GO注释，初步分析了这些蛋白的功能和生理学过程，为进一步研究Jurkat T细胞质膜蛋白的功能奠定了基础。 关 键 词：质膜蛋白质组，Jurkat T细胞，HPLC-CHIP-MS/MS Proteomics analysis of plasma membrane proteins of Jurkat T cells Gan cheng-jun, Tan jiang-lin, Hu jie, Wang xiao-juan, Zhang cui-hua, Zhang xiao-rong, Luo gao-xing, He wei-feng, Wu jun (State Key Laboratory of Trauma，Burns and Combined Injury，Institute of Burns，Southwest Hospital; Chongqing Key laboratory of Proteomics of Diseases, Chongqing 400038,China ) Abstract: Objective To analyze the components of Jurkat T cells plasma membrane protein using one dimensional gel electrophoresis combined with HPLC-CHIP-MS/MS, and the physiological process and function of these proteins by GO(Gene Ontology)annotation for further study of Jurkat T cells membrane proteins. Methods Differential density gradient centrifugation was employed to extract membrane proteins from Jurkat T cells. After SDS, all protein bands were cut off and went through in-gel digestion. Then the obtained peptide fragments were identified by HPLC-CHIP-MS/MS. Bioinformatics was used to analyze the identified proteins. All plasma membrane proteins were picked out and analyzed by GO analysis. Results Total membrane proteins of Jurkat T cells were successfully extracted, and the plasma membrane protein profile was established. After GO annotation, within the 618 membrane proteins identified,