【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0.3μL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。更多还原

【Abstract】 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 O30 O 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.更多还原