色谱

色谱

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蛋白质高效分离两维色谱柱的选择优化

洪广峰1, 高明霞1, 晏国全1, 关霞1, 陶芊1, 张祥民1,2*   

  1. 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 200433
  • 收稿日期:2010-01-05 修回日期:2010-02-01 出版日期:2010-02-28 发布日期:1981-06-25
  • 通讯作者: 张祥民

Optimization of two-dimensional high performance liquid chromatographic columns for highly efficient separation of intact proteins

HONG Guangfeng1, GAO Mingxia1, YAN Guoquan1, GUAN Xia1, TAO Qian1, ZHANG Xiangmin1,2*   

  1. 1. Department of Chemistry, Fudan University, Shanghai 200433, China;2. Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
  • Received:2010-01-05 Revised:2010-02-01 Online:2010-02-28 Published:1981-06-25
  • Contact: ZHANG Xiangmin

PDF

677

被引次数

14

摘要： 为了构建高效的离子交换/反相二维液相色谱(IEC/RPLC)分离平台系统,提高复杂蛋白质样品的分离效率,对色谱柱进行了评价与筛选。通过对实际人肝蛋白质样品的分离效果的比较,选择确定了TSKgel DEAE-5PW弱阴离子交换色谱柱(WAX)作为第一维色谱分离柱；考察了同一规格的10支代表性反相色谱柱(250 mm×4.6 mm, 5 μm, 30 nm, C4、C8或C18),通过评价其对尿嘧啶、硝基苯、萘和芴的分离性能以及对3种标准蛋白质样品的非特异性吸附、对人肝蛋白质样品的WAX馏分的分离效果,最终确定以Jupiter 300 C4反相色谱柱作为第二维色谱分离柱。对两维色谱柱的选择优化为蛋白质高效分离二维液相色谱平台的搭建提供了可靠基础。

关键词: Top-down技术 , 蛋白质, 蛋白质组学, 多维液相色谱

Abstract: In order to optimize two-dimensional liquid chromatographic (2D-LC) columns for highly efficient separation of proteins, several liquid chromatographic columns were investigated and evaluated. Weak anion-exchange (WAX) column was chosen as the first dimension because of its extensive protein separation power. By comparison of different WAX chromatographic columns for human liver protein separation, TSKgel DEAE-5PW column was selected as the first dimension of a 2D-LC system. For the second dimension, ten typical reversed-phase (RP) LC columns (250 mm×4.6 mm, 5 μm, 30 nm) were investigated and evaluated. Their silica based RP stationary phases were butyl (C4), octyl (C8) or octadecyl (C18). To evaluate the retention behavior and non-specific protein adsorption ability of these ten columns, four neutral compounds (uracil, nitrobenzene, naphthalene and fluorene) and three standard proteins (cytochrome C, myoglobin and albumin from chicken egg white) were adopted and separated by RPLC. Meantime, WAX fractions were used to investigate the separation ability of different alkyl-bonded silica stationary phase columns for complex protein samples. By comparison of column separation efficiency, adsorption of intact proteins and sample analysis, Jupiter 300 C4 column was finally employed for its excellent separation ability. Optimization of WAX and RPLC columns offers reliable foundation for the construction of 2D-LC protein separation systems.

Key words: intact protein, multidimensional liquid chromatography, Top-down , proteomics