基质辅助激光解吸电离-飞行时间质谱法检测罕见单糖合成酶

摘要

以VioF(普罗威登斯菌O30中O抗原基因簇内)编码的甲酰基转移酶酶促反应产物为研究对象,采用碰撞诱导解离(CID)负离子模式基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)技术建立简单、高效的罕见单糖合成酶监测方法.本方法首先直接质谱分析03 μL未经色谱分离、除盐处理的酶促反应混合物,随后应用CID串联质谱技术对酶活反应产物进行结构表征,实现酶活反应的快速监测.结果表明:高能CID MALDI-TOF/TOF-MS平台适用于新建克隆的单糖合成酶酶活反应的监测,与现有的监测方法相比,在速度、灵敏度、重现性、自动化和溶剂消耗方面具有绝对优势.%Considering the importance of carbohydrate moieties on infectivity and host mimicry, there is a need to better understand the biosynthetic pathways of these unusual sugars in order to identify key targets involved in bacterial pathogenesis. 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 unusual sugar biosynthesis enzyme activity by using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry with collision-induced dissociation (CID) is demonstrated by VioF (in Providencia alcalifaciens 030 0 antigen gene cluste) enzymatic reaction. The rapid and direct monitoring of the VioF catalyzed reaction was achieved by subjecting a small amount (0.3 μL) of the VioF product mixture to MS analysis without chromatographic separation or desalting steps, and subsequent MS-MS analyses of the product via collision-induced dissociation enabled the structure of the VioF product of enzyme-catalyzed reaction to be determined. The results obtained were in good agreement with that of traditional HPLC monitoring and preparation, as well as NMR structural characterization. Collectively, these data demonstrate that a high-energy CID MALDI-TOF/ TOF MS-based platform is applicable to the facile determination of the enzymatic activity of other newly cloned uncommon sugar biosynthesis enzymes involved in important uncommon deoxythymidine-diphosphate (dTDP)-sugar biosynthesis pathways and offers significant advantages over current methods in terms of speed, sensitivity, reproducibility, automation and reagent costs.