多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩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质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .

Analysis of Mouse Liver Plasma Membrane Proteins by Multidimensional Liquid Chromatography-Tandem Mass Spectrometry

A automated on line nanoflow multidimensional liquid chromatography coupled with tandem mass spectrometry was used to identify mouse liver plasma membrane(PM) proteins separated and enriched by sucrose density gradient centrifugation. A strong cation exchange(SCX) column worked as the first phase and a reversed phase capillary column as the second phase. A pre column was incorporated between the two phases of chromatography .The proteins were extracted from the plasma membrane by detergents assisted solution. After a optimized tryptic digestion of PM proteins, the digested peptide mixture was loaded onto the SCX column, and 10 different concentrations of ammonium acetate solution were injected to elute the ion exchanged peptides respectively. Following desalinisation the eluted peptides were further purified by reversed phase capillary column and online analysed by the coupled tandem mass spectrometry. The acquired MS and MS/MS spectra, after being processed with analytical software ProteinLynx , were utilized to search the Swiss Prot database with Mascot search engine for protein identification . A total of 126 proteins were identified, of which 41 were mouse liver membrane proteins, including membrane assisted proteins and integral membrane proteins with multiple transmembrane domains (TMDs). The results provide fundamental information for the methodology of the mouse liver plasma membrane proteomics and the related database.