质谱学报2011，Vol.32Issue(4)：193-199,7.
糖基转移酶反应的基质辅助激光解吸电离-飞行时间质谱法监测
Monitoring of Glycosyltransferase Reactions by MALDI-TOF Mass Spectrometry
摘要
Abstract
Novel gram-negative bacteria have lipopolysaccharides terminating in repeating oligosaccharides which comprise the O-antigen. The glycosyltransferases (GTs) assembling the Ochain utilize lipid-linked acceptor substrates and nucleotide sugar donor substrates. However, the lack of a rapid and simple method for monitoring glycosyltransf erase activity preclude the elucidation of the function of putative GTs. Mass spectrometry is a rapid, sensitive, and accurate approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study, a simple and efficient assay for glycosyltrans-ferase activity by matrix-assisted laser desorption-ionization (MALDI) mass spectrometry with collision-induced dissociation (CID) was demonstrated by wabD (in E. coli O77 O-an-tigen gene cluste) enzymatic reaction, wfgD (in E. coli O152 O-antigen gene cluste) enzymatic reaction and wfeD (in S. boydii O14 O-antigen gene cluste) enzymatic reaction, respectively. The rapid and direct detection of the enzymatic reaction was achieved by subjecting a small amount (0.3 μL) of the reaction mixture to MS analysis without chromato-graphic separation or desalting steps, and subsequent MS-MS analyses of the product via collision-induced dissociation enabled the structures of products of enzyme-catalyzed reactions to be determined. Collectively, these data demonstrate that CID MALDI-TOF MS based platform is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility, automation and reagent costs.关键词
脂寡糖/糖基转移酶/基质辅助激光解吸离子化-飞行时间质谱( MALDI-TOF MS)Key words
glucosyltransferase/ lipooligosaccharide/ matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS)分类
化学化工引用本文复制引用
周大炜,刘斌,吴俊丽,胡波,齐源远..糖基转移酶反应的基质辅助激光解吸电离-飞行时间质谱法监测[J].质谱学报,2011,32(4):193-199,7.基金项目
南开大学引进人才科研启动资金(No.J02006)资助 （No.J02006）
