人中心体的纯化提取及蛋白质谱分析  

Centrosome isolation and analysis by mass spectrometry-based proteomics

作　　者：钟铠泽[1,2,3] 赵健[2] 袁首道[4] 王庐宇 荆艳洁 郝彤[3] 陈健行[4] 詹启敏[5] 姜伟[3,5] 朱长军[3] 潘丽娜[3] 

机构地区：[1]天津市第一中心医院胸外科,天津300192 [2]山东大学齐鲁医院胸外科,山东济南250012 [3]天津师范大学生命科学学院分子细胞系统生物学重点实验室,天津300387 [4]山东大学癌症研究中心,山东济南250012 [5]中国医学科学院中国协和医科大学分子肿瘤学国家重点实验室,北京100021

出　　处：《山东大学学报（医学版）》2014年第12期54-59,共6页Journal of Shandong University:Health Sciences

基　　金：分子肿瘤学国家重点实验室开放课题(SKL-KF-2013-12);国家自然科学基金(31271485);教育部“新世纪优秀人才支持计划”(NCET-11-1066)

摘　　要：目的提取人类细胞中的中心体,结合蛋白质谱分析,鉴定中心体蛋白组成。方法采用密度梯度离心方法提取K562细胞内中心体。经蛋白免疫印迹鉴定后,对样品进行蛋白质谱分析检测。对已检测出的中心体蛋白分子,应用基因本体(GO)进行系统分析。结果成功提取纯化了人类细胞的中心体,检测出中心体相关蛋白分子γ-Tubulin集聚在特定密度梯度离心分离的细胞裂解液组分中,该组分中无线粒体蛋白VDAC1和核糖体蛋白RPL15。经蛋白质谱分析,发现中心体蛋白组分包括多种细胞质物质运输蛋白、细胞骨架蛋白、膜结合蛋白、蛋白激酶等蛋白分子,涵盖多种细胞内生物信号途径。结论成功提取人细胞内中心体,可为提取纯化不同生长时期细胞中的中心体奠定实验技术基础,结合蛋白质谱分析有助于理解中心体的基本结构和各种功能。Objective To identify the composition of centrosome proteins by human centrosome extraction, purification and protein spectrum analysis. Methods Centrosomes in K562 cells were extracted through density gradient centrifugation. Protein spectrum analysis was carried out on the sample tested by Western blotting. System analysis of all these centrosome proteins were proceeded by Gene Ontology （GO）. Results The human centrosome was successfully extracted and purified. Cell lysis fractions where centrosome-associated protein γ-Tubulin accumulated were detected. There were no mitochondrial protein VDAC1 and ribosomal protein RPL15 in these fractions. The centrosome protein components, including cytoplasm material transport protein, cytoskeleton protein, membrane combining protein, kinase protein and many other protein molecules, covered a variety of the biological message pathways within cells. Conclusion Centrosomes in different cell growth periods can be extracted and purified through this method. Combi- ning protein spectrum analysis, this study is helpful to understand the basic structure and functions of the centosome.

关 键 词：中心体 蛋白质谱分析 蛋白组学 

分 类 号：Q291[生物学—细胞生物学]