大鼠海马细胞质膜的分离及其蛋白质组学分析

Separation of Adult Rat Hippocampal Plasma Membrane and Its Proteomic Analysis

运用差速离心和连续蔗糖密度梯度离心的方法分离纯化成年大鼠海马组织细胞质膜并用Western印迹对质膜样品进行检测.结果表明,在蔗糖密度梯度离心介质中,膜片主要集中于蔗糖浓度为32%～42%的区段(密度约1.13～1.18),其次是20%～25%(密度约1.08～1.10)的区段.39%(密度约1.17)层面上质膜浓度和纯度最高.一维SDSPAGE分离和CapLCMSMS分析后,通过数据库搜寻从大鼠海马组织细胞质膜样品中共鉴定出135种蛋白质,其中质膜蛋白和与膜相关的蛋白质共70种(占51.9%),主要包括Na+K+ATPase、谷氨酸天冬氨酸转运蛋白(glutamateaspartatetransporter)、亲脂素(lipophilin)、GLAST1a等.为研究海马组织细胞的功能和大鼠海马组织细胞质膜蛋白质数据库的建立积累了有价值的资料.

The adult rat hippocampal plasma membrane was purified by differential velocity centrifugation and continuous sucrose density-gradient centrifugation sequentially. Validation of the obtained plasma membrane was performed with Western blotting. The results showed that there were two plasma membrane bands within the gradient matrix, the major in the region of 32% -42% sucrose （density 1.13 - 1.18） and the minor in the region of 20% - 25% sucrose （density 1.08 - 1.10） ; the region of about 39% sucrose （density 1.17） contained highest concentration and purity of plasma membrane. After analysis by SDS-PAGE and CapLC-MS/ MS, totals of 135 proteins of rat hippocampal plasma membrane preparations were identified through database searching. Of the identified proteins, 70 were plasma membrane and membrane-associated proteins （accounting for 51.9 %）, including Na^＋/K^＋ -ATPase, Glutamate/aspartate transporter, Lipophilin, GLAST la and so on. The present experimental results accumulated valuable data for understanding the function of hippocampus and constructing the protein database specific for rat hippocampus.