色谱

色谱

• 研究快报 •    下一篇

微柱液相色谱-电喷雾串联质谱检测多磷酸化肽位点

王晖1,2,段继诚1,2,袁辉明1,2,张丽华1,张维冰1,张玉奎1

  

  1. 1.National Chromatographic R. &A. Center, Dalian Institute of Chemical Physics, the Chinese Academy of Sciences, Dalian 116023, China; 2.Graduate University of Chinese Academy of Sciences, Beijing 100039, China
  • 收稿日期:2007-04-09 修回日期:2007-06-22 出版日期:2007-07-30 发布日期:1985-06-25
  • 通讯作者: 张丽华

Detection of Multi-Phosphopeptide Sites Using Microcolumn High Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

WANG Hui1,2, DUAN Jicheng1,2, YUAN Huiming1,2, ZHANG Lihua1, ZHANG Weibing1, ZHANG Yukui1   

  1. 1.National Chromatographic R. &A. Center, Dalian Institute of Chemical Physics, the Chinese Academy of Sciences, Dalian 116023, China; 2.Graduate University of Chinese Academy of Sciences, Beijing 100039, China
  • Received:2007-04-09 Revised:2007-06-22 Online:2007-07-30 Published:1985-06-25

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摘要：

基于碱性磷酸酶的去磷酸化作用，发展了一种可改善多磷酸化肽电喷雾质谱检测效果的技术。将β-酪蛋白酶解产物用TiO2柱富集后，用碱性磷酸酶进行处理，并经微柱液相色谱分离后采用串联质谱进行鉴定。通过谱图中存在相对分子质量与根据氨基酸序列预计的单磷酸化肽相差80 的色谱峰，可以证实样品中含有单磷酸化肽。此外，经碱性磷酸酶处理后的样品的色谱峰数目的增加，说明样品中可能存在多磷酸化肽段。通过控制去磷酸化反应的程度，使四磷酸化肽的部分磷酸基团被去除，从而可以推断其中3 个磷酸化位点可能处于氨基酸残基序列的第17、18和19位。

关键词: 电喷雾串联质谱, 多磷酸化肽, 碱性磷酸酶 , 磷酸化位点, 微柱液相色谱

Abstract: A novel detection method for the analysis of multi-phosphopeptides using microcolumn high performance liquid chromatography-electrospray ionization tandem mass spectrometry (μHPLC-ESI-MS/MS) was proposed by the dephosphorylation treatment of alkaline phosphatase (AP). After the selective enrichment by a microcolumn packed with TiO2, phosphopeptides from the tryptic digests of β-casein were dephosphorylated by AP. Through the removal of phosphate groups, the detection of multi-phosphopeptides according to the non-phosphorylated ones was achieved by ESI-MS/MS. By comparing the chromatograms before and after the AP treatment, mono-phosphopeptides were identified based on the relative molecular mass (Mr) difference of 80. Furthermore, since more peaks appeared after the treatment, the existence of multi-phosphopeptides was proven. By controlling the treatment procedure, the partial dephosphorylation of multi-phosphopeptides was performed, and the multi-phosphorylated sites of the digest of β-casein were found to be on serine residues at the possible sites of 17, 18 and 19.

Key words: alkaline phosphatase , electrospray ionization tandem mass spectrometry (ESI-MS/MS), multi-phosphopeptide, phosphorylation site, microcolumn high performance liquid chromatography (μHPLC)