用于人血浆中蛋白质分离的在线阵列式二维常规柱液相色谱系统的建立  

Development of online conventional array-based twodimensional liquid chromatographic system for proteins separation in human plasma

作　　者：黄志[1,2] 洪广峰[1] 高明霞[1] 张祥民[1,2] 

机构地区：[1]复旦大学化学系,上海200433 [2]复旦大学生物医学研究院,上海200032

出　　处：《色谱》2014年第4期343-348,共6页Chinese Journal of Chromatography

基　　金：国家高技术研究发展计划项目(2012AA020202);科技部基础研究计划项目(2012CB910604);国家自然科学基金项目(21027002;21175026;21275034)

摘　　要：构建了一种在线阵列式二维常规柱液相色谱系统,并将其应用于分离血浆中的完整蛋白质。该系统以1根强阴离子交换柱作为第一维分离柱,8根阵列式反相色谱柱作为第二维分离柱。强阴离子交换柱分离的馏分通过十通阀被依次转移到第二维预柱上并得到保留富集,随后第二维流动相通过分流器同时将预柱上的蛋白质反冲至分析柱上进行分离。二维之间以及第二维阵列色谱柱之间均相互独立,从而可以提高系统分离的通量和总峰容量。采用该系统对血浆中的蛋白质进行了完整蛋白质水平上的分离。该系统具有高通量和高分辨率的特点,为血浆样品中高丰度蛋白质的去除以及血浆样品的深入研究提供了一种有效的手段。Human plasma is one of the proteins-containing samples most difficult to character-ize on account of the wide dynamic concentration range of its intact proteins. Herein,we devel-oped a high-throughput conventional array-based two-dimensional liquid chromatographic sys-tem for proteins separation in human plasma in online mode. In the system,a conventional strong-anion exchange chromatographic column was used as the first separation dimension and eight parallel conventional reversed-phase liquid chromatographic columns were integrated as the second separation dimension. The fractions from the first dimension were sequentially trans-ferred into the corresponding reversed-phase liquid chromatographic precolumns for retention and enrichment using a 10-port electrically actuated multi-position valve. The second dimen-sional solvent flow was directly and identically split into 8 channels. The fractions were concur-rently back-flushed from the precolumns into the 8 conventional RP columns and were separa-ted simultaneously. An 8-channel fraction collector was refitted to collect the reversed-phase liquid chromatographic fractions for further investigation. Bicinchoninic acid （ BCA ） dyeing solution was conveniently used for high-abundance protein location. Two separation dimensions were relatively independent parts,as well as each channel of the second dimensional array sep-aration. Therefore,the new system could improve the separation throughput and total peak capacity. The system was successfully applied for the separation of human plasma intact pro-teins. The results indicated the established system is an effective method for removing high abundance proteins in plasma and in-depth research in plasma proteomics.

关 键 词：阵列二维色谱 在线 蛋白质 血浆 蛋白质组学 

分 类 号：O658[理学—分析化学]