微柱液相色谱-电喷雾串联质谱检测多磷酸化肽位点  

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

作　　者：王晖[1] 段继诚[1] 袁辉明[1] 张丽华[1] 张维冰[1] 张玉奎[1] 

机构地区：[1]中国科学院大连化学物理研究所国家色谱研究分析中心

出　　处：《色谱》2007年第4期453-456,共4页Chinese Journal of Chromatography

基　　金：中国科学院知识创新工程重要方向项目(No.KJCX2.YW.H09);国家自然科学基金重点基金(No.20435020)资助项目

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。A novel detection method for the analysis of multi-phosphopeptides using microcolumn high performance liquid chromatography-electrospray ionization tandem mass spectrometry （μLHPLC-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-phospho- rylated sites of the digest of β-casein were found to be on serine residues at the possible sites of 17. 18 and 19.

关 键 词：微柱液相色谱 电喷雾串联质谱 多磷酸化肽 磷酸化位点 碱性磷酸酶 

分 类 号：O658[理学—分析化学]