在线微酶反应器-微柱液相色谱-电喷雾质谱用于蛋白质快速分析  

On-line Microenzymatic Reactor-Micro Column HPLC-ESI-MS/MS for Rapid Protein Analysis

作　　者：袁辉明[1,2] 张丽华[1] 梁振[1] 张玉奎[1,3] 

机构地区：[1]中国科学院大连化学物理研究所国家色谱研究分析中心,大连116023 [2]中国科学院研究生院,北京100039 [3]中国科学院

出　　处：《化学通报》2009年第5期427-432,共6页Chemistry

基　　金：国家自然科学基金重点项目(20435020);国家支撑计划项目(2006BAK03A00)资助

摘　　要：采用带有环氧基的聚丙烯酸酯基质微球实现了胰蛋白酶的固定化,并制备了填充式微酶反应器。以细胞色素C为底物,考察了微酶反应器的性能。与自由溶液酶解相比,细胞色素C在微酶反应器上的酶解时间可以缩短到18s以内,速度是自由溶液酶解的2400倍。在此基础上,构建了在线微酶反应器-微柱液相色谱-电喷雾质谱联用平台,并采用4种标准蛋白混合物进行了评价。结果表明,4种蛋白均得到了较好的酶解和鉴定结果。此外,该平台还被应用于鼠肝提取蛋白的分析,共鉴定出138种蛋白,其中匹配两个或两个以上肽段的蛋白质有31个。Abstract Trypsin was immobilized on polyacrylic ester particles with epoxy groups, and an immobilized trypsin microreactor was prepared by packing such polymer particles into a PEEK column （0.5mm i. d× 35mm）. With cytochrome c as a model sample, the performance of the micrnreactor was evaluated. Compared with in-solution digestion protocol, high efficient digestion was achieved within 18s by the microreactor, with the digestion speed improved by 2400 times. Based on such a microreactor, an integrated platform of on-column digestion, peptides separation and MS/MS identification was constructed, and evaluated by the analysis of four-protein mixture. Furthermore, the platform was applied for the analysis of proteins extracted from mouse liver, and 138 proteins were identified, of which 31 proteins were identified with two or more than two unique peptides matched. Keywords Enzymatic microreaetor, μHPLC, ESI-MS/MS, Protein, On-line analysis

关 键 词：微酶反应器 微柱液相色谱 电喷雾质谱 蛋白质 在线分析 

分 类 号：Q51-3[生物学—生物化学]