Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry
Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry
复制标题
DOI：
10.1002/jms.593
复制
发表时间：
2004-02-01
影响因子：
2.3
通讯作者：
Smith, RD
中科院分区：
文献类型：
--
作者：
Kim, J;Camp, DG;Smith, RD
与较低的磷酸化状态（例如来自牛β-酪蛋白的胰蛋白酶单磷酸肽RELEELNVPGEIVEpSLpSpSpSEESITR）相比，多磷酸化肽（例如来自牛β-酪蛋白的胰蛋白酶消化的四磷酸肽RELEELNVPGEIVEpSLpSpSpSEESITR）的特异性检测对于液相色谱质谱（LC/MS）分析来说经常是有问题的，这是由于它们对暴露表面的吸附的高亲和力。我们观察到磷酸（0.1-1.0%）添加到样品溶液中的磷酸肽的整体检测的增强，灵敏度增加10倍，确定为两个胰蛋白酶磷酸肽的检测，也是一个显着的改进，在检测的四磷酸肽。使用毛细管LC与离子阱串联MS进行检测和鉴定，可实现的检测限为50 fmol和50 pmol的单磷酸肽和四磷酸肽，分别。磷酸被认为充当二氧化硅毛细管表面和C-18键合的固定相二氧化硅表面上的可用硅烷醇基团的阻断剂。版权所有（C）2004约翰威利父子有限公司。
In contrast to lower phosphorylation states (e.g. the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine beta-casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine P-casein) has often been problematic for liquid chromatographic mass spectrometric (LC/MS) analysis owing to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides on addition of phosphoric acid (0.1-1.0%) to the sample solution; a 10-fold increase in sensitivity was determined for the detection of two tryptic phosphopeptides and also a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with ion trap tandem MS for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C-18-bonded stationary phase silica surface. Copyright (C) 2004 John Wiley Sons, Ltd.