亲水相互作用色谱结合串联质谱多重碎裂模式在完整糖肽研究中的应用  

Characterization of Intact Glycopeptides by a Combination of Hydrophilic Interaction Liquid Chromatography and Multiple Fragmentation Tandem Mass Spectrometry

作　　者：江静[1,2] 应万涛[2] 钱小红[2] 

机构地区：[1]中国人民解放军总医院,军医进修学院,北京100039 [2]蛋白质组学国家重点实验室,北京蛋白质组研究中心,解放军医学院,北京102206

出　　处：《分析化学》2014年第2期159-165,共7页Chinese Journal of Analytical Chemistry

基　　金：国家高技术研究发展计划(No.2012AA020203);国际科技合作项目(No.2011DFB30370);北京市科技新星计划(No.Z121107002512014)资助~~

摘　　要：蛋白质的糖基化修饰在生理及病理过程中发挥着重要作用。由于技术条件的限制,传统的糖蛋白分析方法中,通常分别鉴定蛋白质和糖链两者的结构,而忽略了蛋白质与糖链的连接关系。本研究旨在以完整糖肽作为检测对象,对糖肽中肽段序列和糖链结构的同步解析。采用亲水相互作用色谱对完整糖肽进行分离纯化,联合生物质谱中碰撞诱导解离(CID)、高能诱导解离(HCD)、电子转移解离(ETD)等裂解模式,对完整糖肽的糖基化位点、糖链结构、肽段序列等进行全方面的解析。结果表明,亲水相互作用色谱中样品与填料比1∶50可有效富集糖肽,采用30%CID能量,主要产生糖苷键断裂的碎片,为糖链的结构组成分析提供了线索;采用25%HCD能量,在低分子量区域提供了糖链的特征离子信息,并产生明确的糖肽Y1特征离子;ETD保留了完整的修饰基团而产生肽段骨架的断裂,可以提供有效的肽段序列信息。本研究结合亲水相互作用色谱与质谱仪中的多种碎裂方式,为完整糖肽的结构解析提供了一种快速、有效的研究方案。Glycosylation of proteins plays a significant role in physiological and pathological processes. The general strategy to characterize the glycoprotein is to release the glycans from proteins, and then analyze them separately. However, the traditional methods suffer from inherent limitations since the relationship between proteins and glycans is neglected. Here we developed an integrated approach for intact glycopeptides analysis. Glycopeptides were first enriched selectively by home-made hydrophilic interaction liquid chromatography （HILC） tips, and then analyzed by mass spectrometry. We applied multiple fragmentation methods including collision-induced dissociation （CID）, high energy collisional dissociation （HCD） and electron transfer dissociation （ETD） for comprehensive characterization of glycosylation. The results showed that the best ratio of sample vs material for glycopeptide enrichment was 1：50; CID with 35% collision energy cleaves glycosidic bonds, which was useful for glycan composition identification; HCD with 25% collision energy provided low mass diagnostic ions specific to glycan fragmentation, and also the specific Y1 fragment ion of glycopeptide; ETD cleaved peptide backbone, yielded peptide sequence and helps to assign glycosites. This work indicates that HILIC enrichment and multiple fragmentation techniques may be an effective way to characterize glycopeptides for the simultaneous elucidation of peptide sequence, glycosite and glycan structures.

关 键 词：亲水作用色谱 串联质谱 完整糖肽 碰撞诱导解离 高能诱导解离 电子转移解离 

分 类 号：Q51[生物学—生物化学] O657.63[理学—分析化学]