通过生物素化标记分析细胞膜亚蛋白质组

细胞膜在许多基本生物学作用过程中扮演着重要角色，比如细胞-细胞相互作用、信号转导和物质运输.分析不同生理或病理状态细胞的膜蛋白的表达变化，有助于了解这些生物学过程以及发现新的诊断、治疗靶位.成功地进行膜蛋白分析最重要的是获得足够浓度与纯度的膜蛋白.这里报告一种分离高纯度膜蛋白的方法，主要包括三个步骤：（1）生物素化活细胞表面的膜蛋白，（2）链亲和素琼脂糖亲和吸附，（3）强烈的清洗条件去除非特异吸 附的胞质蛋白.最后用化学方法洗脱生物素标记的膜蛋白进行双向电泳分离分析.用该方法 制备了HeLa细胞的膜蛋白.免疫印迹结果表明，生物素标记的膜蛋白基本上都是低丰度蛋白. 这样制备得到的膜蛋白双向电泳分离出2 400多个蛋白质点，而且大多数点的亮度相近，分 布得相对分散没有聚集成群.这种图谱非常便于比较分析找到差异蛋白.多次重复制备膜蛋白 、电泳表明该方法的重复性和稳定性很好.

Analysis of Cell Membrane Subproteome by Biotinylation of Proteins

Plasma membrane plays important roles in many basic biological processes such as cell-cell interaction, signal transduction and material transport. Analysing expression pattern of membrane protiens of cells in different conditions will increase the understanding these processes and discovering new protein targets for design of diagnostic or therapeutic drugs. Isolating membrane proteins with high concentration and purity is crucial to successful proteomic analysis. A method for preparation of membrane proteins is reported here, which involves (1) biotinylation of cell surface proteins in viable cells, (2) affinity enrichment using strepavidin sepharose, (3) depletion of nonspecifically absorbed cytosolic proteins by harsh washes. The biotinylated membrane proteins are then extracted and subjected to two dimensional electrophoresis separation. Membrane proteins from HeLa cell line were prepared with this method. Western blotting analysis showed that most of biotinylated membrane proteins are belong to those of low abundance. There are about 2 400 spots on the 2-D profile of biotinylated membrane proteins, most spots with similar abundance and distributed separately. Such profile is suitable for comparative analysis.