色谱

色谱

• 研究论文 • 上一篇    下一篇

基于强阳离子交换色谱分离的磷酸化肽段富集策略

隋少卉1,2,王京兰1,卢庄1,蔡耘1,张养军1,蔚文峰1,钱小红1

  

  1. 1.State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China; 2.Institute of Chemical Defence, Beijing 102205, China
  • 收稿日期:2007-11-09 修回日期:2008-01-03 出版日期:2008-03-30 发布日期:1984-06-25
  • 通讯作者: 钱小红

Phosphopeptide enrichment strategy based on strong cation exchange chromatography

SUI Shaohui1,2, WANG Jinglan1, LU Zhuang1, CAI Yun1, ZHANG Yangjun1, YU Wenfeng1, QIAN Xiaohong1

  

  1. 1.State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China; 2.Institute of Chemical Defence, Beijing 102205, China
  • Received:2007-11-09 Revised:2008-01-03 Online:2008-03-30 Published:1984-06-25
  • Contact: Xiao-Hong Qian

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摘要：

为了在短时间内获得相对含量高的磷酸化肽段，以标准磷酸化蛋白质为模型对强阳离子交换色谱（SCX）分离磷酸化肽段体系的缓冲溶液和梯度设置进行了研究，并用酵母酶切肽段混合物考察了该路线在较复杂的样品中的应用。实验结果表明优化后的体系能够在30 min内分离出磷酸肽段，而且非磷酸化肽段的干扰很少，这样便相对提高了磷酸化肽段在质谱仪中的响应强度，重要的是该体系可以对复杂样品进行很好的分离。这说明SCX用于规模化磷酸化肽段富集的策略是可行的。本研究为磷酸化蛋白质组学规模化分析提供了实用技术。

关键词: 蛋白质组学 , 富集, 磷酸化, 强阳离子交换色谱, 位点

Abstract:

The routine analysis of phosphoproteomics needs effective and specific enrichment methods. The strong cation exchange chromatography (SCX) separation conditions, including the buffer solution and the separation gradient, were studied to enrich the phosphopeptides in a short time. Firstly, the tryptic peptides of standard protein mixtures (bovine α-casein and β-casein, chicken egg ovalbumin) were used to optimize the SCX strategy. Then, this method was applied to the complicated samples, the tryptic peptides from yeast. The experimental results showed that the phosphopeptides were eluted before 30 min in the optimized SCX systems with less interference from nonphosphopeptides. Phosphopeptides were successfully separated in a short time, and the ion signals corresponding to phosphopeptides were accordingly increased during mass spectrometric analysis. It was indicated that the optimized SCX separation system could be used as a simple enrichment method for the separation of phosphopeptides, especially for phosphopeptides in the complex real samples, in a short time, and to increase the relative abundance of phosphopeptides in the detection fraction. This study provides the pragmatic technique for phosphoproteomics analysis on a large-scale.

Key words: enrichment, proteomics , sites, strong cation exchange chromatography (SCX), phosphorylation