【作者】 黄纯翠； 孙世伟； 王耀君； 刘亚名； 周巾煜； 杨飞； 高枫； 陈润生； 柴文刚； 卜东波； 李岩；

【Author】 Chuncui HUANG;Shiwei SUN;Yaojun WANG;Yaming LIU;Fei YANG;Jingwei ZHANG;Feng GAO;Jinyu ZHOU;Runsheng CHEN;Wengang CHAI;Dongbo BU;Yan LI;Institute of Biophysics, Chinese Academy of Sciences;Key Lab of Intelligent Information Processing, Institute of Computing Technology;University of Chinese Academy of Sciences;Imperical College London;

【机构】 中国科学院生物物理研究所； 中国科学院计算技术研究所； 帝国理工学院；

【摘要】 生物糖链对蛋白质构象、分子识别、细胞增殖和分化等过程具有重要影响,因而糖链分支结构的准确鉴定对于功能糖组学具有重要的意义。多级质谱技术可以对糖链碎片进行多级断裂,反映出碎片的不同层次的结构信息,因此成为糖链结构鉴定的一项重要技术。目前,多级质谱技术鉴定糖链结构面临两个困难:一是如何准确且有效选择母离子碎片产生下级质谱谱图;二是如何通过众多的多级质谱图解析出糖链的分支结构。为了解决上述两个难题,本文首先提出一种自动选择最具结构信息的碎片产生多级质谱谱图的方法,然后基于多级质谱谱图信息鉴定出糖链的分支结构,最后应用建立的方法鉴定生物样品中的糖链分支结构。实验结果表明,利用建立的多级质谱方法可准确鉴定标准糖链的分支结构,并且利用此多级质谱方法可实现糖链异构体的鉴定,最后新建的多级质谱方法能够准确鉴定糖蛋白和生物样品中糖链的分支结构。因此,本研究建立的多级质谱方法对于自动化鉴定糖链结构具有重要推动作用,已经成功实现高通量的蛋白质药物与复杂血液样品中的糖链结构鉴定。更多还原

【Abstract】 Glycans play important roles in a variety of biological processes such as protein conformation, molecular recognition, cell proliferation and differentiation [1]. Their activities are closely related to the fine details of their structures. Unlike the simple linear chains of proteins, branching is a unique feature of glycan structures, making their identification extremely challenging. Tandem mass spectrometry with collisioninduced dissociation(MS~2) has been used successfully in glycan sequence determination [2]. Glycosidic bond cleavage with B-/Y-and C-/Z-ions affords sequence and branching pattern information while cross-ring fragmentation with A-/X-type ions may occur, although frequently of low abundances, to indicate linkage positions [3]. However, MS~2 is not always sufficient and multiple-stage mass spectrometry(MS~n , n>2) is frequently required to provide detailed structural information. MS~n is a promising way to assign individual glycan structures as it can produce multiple-level fragments of a glycan through consecutive fragmentations.The major difficulty for successful application of MS~n and thereafter automated highthroughput glycan structural identification is precursor-ion selection. Unlike MS~2,sequential MS~n requires careful selection of fragment ions as precursors for the next stage of product-ion scanning. Widely-used strategies are either manual selection by experienced experts or simply selecting the most intense peaks. Manual selection requires considerable expertise and time, whereas product-ion spectrum generated by the most intense peak may not be structurally informative. The concept of‘intelligent precursor selection’ to guide MS~n experiments has been described [4] but a systematic approach for achieving is lacking.In the present communication, we propose an approach for glycan identification using an intelligent precursor-ion selection(GIPS) strategy to guide MS~n experiments,and elucidate glycan branching patterns based on the generated mass spectra. We demonstrate its successful application to identification of a variety of N-glycans and secreted oligosaccharides. Complex samples such as human serum N-glycans were identified using MS~n and GIPS strategy, and detailed branching patterns were provided. The proposed MS~n methodology and GIPS strategy represent a major step forward towards high-throughput and automated glycan omics.更多还原