肝癌细胞线粒体蛋白的MALDI-TOF质谱鉴定方法研究  

MALDI-TOF MS identification of protein of mitochondria from cultured hepatoma cells

作　　者：蒋代凤[1] 邱宗荫[1] 李兴[1] 黄开顺[2] 滕永真[2] 张晓萍[2] 

机构地区：[1]重庆医科大学医学检验系实验诊断教研室,重庆400016 [2]重庆医科大学基础医学研究所,重庆400016

出　　处：《重庆医科大学学报》2005年第4期485-488,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金项目(No:30476021);教育部博士点基金项目(教技发中心函[2004]166号)

摘　　要：目的:探索胶上蛋白原位酶切、肽质量指纹图(PMF)分析以及数据库检索方法的优化条件,建立适合于亚细胞比较蛋白质组学研究的质谱鉴定策略。方法:从考马斯亮蓝染色的2-DE胶上取下牛血清白蛋白(BSA)标准蛋白进行胶内原位酶切,与基质(CHCA)混匀后进行MALDI-TOF MS分析;对来源于人肝细胞癌细胞株QGY-7703线粒体的差异表达的候选蛋白点L9,按照通过BSA标准蛋白优化的条件进行MALDI-TOF质谱鉴定;其肽质量指纹图(PMF)经MS-Fit检索。结果:经数据库检索,BSA的肽质量指纹图(PMF)匹配肽质量数为10/11,序列覆盖率为19%,说明实验条件完全可信。候选蛋白点L9的PMF数据经数据库检索后,排名前两位的蛋白均为OXCT(3-oxoacid CoA transferase 1 precursor),其匹配肽段为9/13,序列覆盖率为24%;且OXCT的理论分子量(56kda),理论等电点(7.1),与胶上L9蛋白的位置相符。故候选蛋白点L9确认为OXCT(3-oxoacid CoA transferase 1 precursor)。结论:本文所确定的鉴定策略适用于线粒体比较蛋白质组学的研究。Objective：To explore the optimal conditions associated with proteolytic digestion in 2 - DE gel, PMF analysis and database inquiry, and to construct a mass identification strategy in research for mitochondrial comparative proteomics. Methods ：The BSA standard protein was incised from 2 DE gel by Coomassic Brilliant Blue dyeing and was mixed with matrix （CHCA）, then analyzed by MALDI-TOF MS. The differentially expressed mitochondria protein L9, an unknown protein obtained from hepatoma cells QGY-7703, was identified by condition with BSA optimizing. The PMF was inquired by MS Fit database. Results ： The BSA was identified in Masses Matched number 10/11 and Sequence Overcast 19% of PMF, which proved to the reliability of experimental conditions. By database Inquiry about PMF from L9 protein, OXCT（3 -oxoacid CoA transferase 1 precursor）was identified with Masses Matched number 9/13 and Sequence Overcast 24 %. The OXCT was in accord with position on gel. Conclusion ：This method is applicable to research for mitochondrial comparative protcomics.

关 键 词：线粒体 蛋白质组学 肽质量指纹图(PMF) 基质辅助激光解吸离子化-飞行时间质谱(MALDI-TOF MASS) 二维电泳(2-DE) 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]