大鼠海马组织线粒体的蛋白质组学分析方法的建立

为了建立蛋白质组学技术平台用于分离分析大鼠海马组织线粒体的蛋白质组成,选用成年雄性Wistar大鼠使用差速离心法分离提纯大鼠海马组织线粒体,经透射电子显微镜观察分析、免疫印迹检测细胞器标志物蛋白含量后确定线粒体提纯物的质量;将线粒体制备成蛋白样品,经双向电泳分离、染色、扫描获得二维蛋白斑点图后,切取含蛋白斑点的胶块处理后进行基质辅助的飞行时间生物质谱分析,所得数据经检索匹配识别出蛋白信息。获得的大鼠海马组织线粒体提纯物在透射电子显微镜下呈现为大量形态完整、典型的线粒体,夹杂少量其它成分和线粒体碎片;免疫印迹结果显示线粒体提纯物蛋白中含有大量稳定表达的线粒体标志蛋白COX-IV;线粒体提纯物中没有检测到细胞核标志蛋白histone-1。双向电泳分离得到的二维蛋白斑点图谱匹配率为77.54%±5.51%,并成功鉴定识别出胶中的线粒体蛋白丙酮酸脱氢酶alpha亚基。上述结果表明已经成功建立了缺血敏感脑区海马组织的线粒体蛋白质组学分析方法,所得线粒体纯度高、蛋白样品重复性高、电泳分离效果好、与生物质谱鉴定技术匹配良好,为开展脑缺血的线粒体蛋白质组学研究打下了基础。

Establishment of the proteomic platform for analysis of rat hippocampal mitochondrial proteome

In order to set up proteomic technical platform for analysis of rat hippocampal mitochondrial proteome,male adult Wistar rats were used.Rat hippocampal mitochondria was separated and purified from tissue by sequential centrifugation,and then identified by transmission electron microscopy and immunoblot to check the quality of purification.Protein samples prepared from purified mitochondria were separated by two-dimensional gel electrophoresis(2DE),gels were stained and then gel images of protein spots were acquired after scanning.Gel pieces containing protein spots were excised.Proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS),and then identified by database search and match.Purified rat hippocampal mitochondria was shown to be a majority of typical mitochondria of intact shape,with few impurity and fragments of mitochondria,under transmission electron microscope.Purified mitochondrial protein samples have stable expression of a large quantity of mitochondrial marker COX-IV by immunoblot.Nucleic marker histone-1 was not detected in protein samples.The two-dimensional gel images of mitochondrial protein have the match ratio of 77.54%±5.51%.Mitochondrial protein pyruvic dehydrogenase alpha unit was identified successfully from gels.In conclusion,proteomic platform to analyze mitochondrial proteins in rat hippocampus,which is susceptible to ischemia,has been set up successfully.Isolated mitochondria from rat hippocampus was pure and suitable to be separated by 2DE.2DE gels of good reproducibility matched mass spectrometry for further research about brain ischemia.