SDS-聚丙烯酰氨凝胶电泳与液质联用技术分离和鉴定小鼠巨噬细胞膜蛋白

用膜蛋白分离试剂盒提取巨噬细胞膜蛋白，然后用SDS-聚丙烯酰氨凝胶电泳进行分离。将每个泳道平均切成8份，合并两个泳道同样位置的胶条，分别进行胶内酶解。酶解得到的多肽经脱盐后进入毛细管反相柱进行反相分离，分离后的肽段直接进入电喷离子源质谱仪进行一级和二级质谱分析。质谱数据用SEQUEST软件对小鼠IPI蛋白数据库进行检索，得到一个含有1000多种蛋白的名单，其中包括458种经GOA注释的膜蛋白。对膜蛋白部分进一步分析发现，其中包括CD11b、TNF-a、F4/80、CD14、CD18、CD86、CD44、CD16、Toll样受体等已知表达在巨噬细胞表面的蛋白分子，还包括另外13种CD分子和18种Ras相关GTPase，除了这些已知蛋白之外，还鉴定出若干新蛋白分子，为进一步深入研究巨噬细胞生物学功能提供了目标分子。

Profiling Membrane Proteome of Macrophages by One-dimensional PAGE and Liquid Chromatography-Tandem Mass Spectrometry

Macrophages are involved in many important biological processes and membrane proteins are the key effector molecules for their functions. However, membrane proteins are difficult to analyze by 2-DE based method because of their intrinsic tendency to self-aggregate during the first dimension separation (IEF). To circumvent the obstacle hampering membrane protein analysis, we combined one-dimensional SDS-PAGE with capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using this technique, we identified 458 GO annotated membrane proteins with extremely high confidence, including most known markers of peritoneal macrophages (e.g., CD11b, F4/80, CD14, CD18, CD86, CD44, CD16 and Toll-like receptor). Thirteen other CD antigens and 18 Ras-related small GTPase were also identified. In addition to those known macrophage membrane proteins, a significant number of novel proteins have also been identified. This research provides a valuable data set of macrophage membrane proteins, thus allowing for more comprehensive study of membrane proteins and a better understanding of the function mechanisms of macrophages in many biological processes.