色谱

色谱

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去除血浆中高丰度蛋白质的二维液相色谱体系的建立

朱绍春1,2, 张学洋1, 高明霞1, 晏国全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 200032
  • 收稿日期:2011-07-15 修回日期:2011-07-22 出版日期:2011-09-28 发布日期:2011-10-28
  • 通讯作者: 张祥民,教授,博士生导师,研究方向为蛋白质多维分离方法及其与生物质谱联用技术. Tel: (021)65643983
  • 基金资助:

    国家重点基础研究发展规划(“973”)项目(No. 2007CB914100)、国家自然科学基金项目(No. 21027002)和上海市重点学科建设项目(B109)

Construction of a two-dimensional liquid chromatography separation system for high abundance proteins depletion in human plasma

ZHU Shaochun1,2, ZHANG Xueyang1, GAO Mingxia1, YAN Guoquan1, ZHANG Xiangmin1,2*   

  1. 1. Department of Chemistry, Fudan University, Shanghai 200433, China; 2. Institute of Biomedical Science, Fudan University, Shanghai 200032, China
  • Received:2011-07-15 Revised:2011-07-22 Online:2011-09-28 Published:2011-10-28

PDF

519

被引次数

7

摘要： 血浆中高丰度蛋白质的存在严重干扰低丰度蛋白质的检测,是困扰血浆蛋白质组学研究的技术瓶颈之一。针对这一热点问题,建立了一种二维液相色谱(强阴离子交换色谱-反相高效液相色谱)分离系统,对血浆中的高丰度蛋白质进行了色谱定位并进行去除。选择TSKgel SuperQ-5PW为第一维色谱分离柱,第二维色谱分离采用Jupiter C4柱,对第一维的馏分进行进一步的分离。通过梯度优化,血浆样品经过二维系统得到充分分离。第二维分离过程中从紫外信号强度高(215 nm,大于20 mAU)的峰中选择10个峰,利用液相色谱-串联质谱鉴定出32种高丰度蛋白质,包括人血清白蛋白、免疫球蛋白G等高丰度蛋白质。该体系为血浆中更多高丰度蛋白质的去除以及血浆蛋白质组学的更深入研究提供了重要思路。

关键词: 蛋白质组学, 二维液相色谱, 高丰度蛋白质, 血浆

Abstract: High abundance proteins existing in human plasma severely impede the detection of low abundance proteins. This is one of the most difficult problems encountered in plasma proteomics research. We developed a two-dimensional liquid chromatography system with strong anion exchange chromatography-reversed-phase liquid chromatography (SAX-RPLC) for the extensive separation of plasma proteins and selective depletion of high abundance proteins. TSKgel SuperQ-5PW was selected as the first dimensional separation column for crude human plasma fractionation and Jupiter C4 column was selected as the second dimensional separation column. Separation gradients of the two-dimensional liquid chromatography system were optimized to ensure an extensive separation of plasma proteins. Ten peaks with high signal intensities (＞20 mAU) at 215 nm during the second dimensional separation were collected and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a result, 32 proteins, all of which were reported to be high abundance proteins in plasma, including human serum albumin (HSA), immunoglobulin G (IgG) and so on were successfully identified. This system provides an effective method for future depletion of more high abundance proteins and in-depth research in human plasma proteomics.

Key words: high abundance protein, plasma, two dimensional liquid chromatography, proteomic