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新型 LC-MS² 产物依赖型平行数据采集功能和数据分析工作流程,用于测序和鉴定完整糖肽。

Novel LC-MS² product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides.

机构信息

Institute of Biological Chemistry, Academia Sinica , 128, Academia Road Sec 2, Nankang, Taipei, 11529, Taiwan.

出版信息

Anal Chem. 2014 Jun 3;86(11):5478-86. doi: 10.1021/ac500945m. Epub 2014 May 13.

Abstract

Data dependent acquisition (DDA) of higher collision energy dissociation (HCD)-MS(2) followed by electron transfer dissociation (ETD)-MS(2) upon detection of glycan-specific oxonium is one of the better approaches in current LC-MS(2) analysis of intact glycopeptides. Although impressive numbers of glycopeptide identification by a direct database search have been reported, false positives remained high and difficult to determine. Even in cases when the peptide backbones were correctly identified, the exact glycan moieties were often erroneously assigned. Any attempt to fit the best glycosyl composition match by mass only is problematic particularly when the correct monoisotopic precursor cannot be determined unambiguously. Taking advantage of a new trihybrid Orbitrap configuration, we experimented with adding in a parallel ion trap collision induced dissociation (CID)-MS(2) data acquisition to the original HCD-product dependent (pd)-ETD function. We demonstrated the feasibility and advantage of identifying the peptide core ion directly from edited HCD-MS(2) data as an easy way to reduce false positives without compromising much sensitivity in intact glycopeptide positive spectrum matches. Importantly, the additional CID-MS(2) data allows one to validate the glycan assignment and provides insight into possible glycan modifications. Moreover, it is a viable alternative to deduce the glycopeptide backbone particularly in cases when the peptide backbone cannot be identified by ETD/HCD. The novel HCD-pd-CID/ETD workflow combines the best possible decision tree dependent MS(2) data acquisition modes currently available for glycoproteomics within a rapid Top Speed DDA duty cycle. Additional informatics can conceivably be developed to mine and integrate the rich information contained within for simultaneous N- and O-glycopeptide analysis.

摘要

在检测到糖基特异性氧鎓离子后,采用更高碰撞能量解离(HCD)-MS(2)进行数据依赖采集(DDA),然后进行电子转移解离(ETD)-MS(2),这是当前 LC-MS(2)分析完整糖肽的较好方法之一。尽管通过直接数据库搜索已经报道了令人印象深刻的糖肽鉴定数量,但仍然存在很高的假阳性率,且难以确定。即使在肽骨架被正确鉴定的情况下,糖基部分也经常被错误地分配。仅通过质量拟合最佳糖基组成匹配的任何尝试都是有问题的,特别是当无法明确确定正确的单同位素前体时。利用新的三杂交轨道阱配置,我们尝试在原始 HCD-产物依赖(pd)-ETD 功能中添加平行离子阱碰撞诱导解离(CID)-MS(2)数据采集。我们证明了从编辑后的 HCD-MS(2)数据中直接鉴定肽核心离子的可行性和优势,这是一种减少假阳性而又不影响完整糖肽阳性谱匹配灵敏度的简便方法。重要的是,额外的 CID-MS(2)数据可以验证糖基分配,并深入了解可能的糖基修饰。此外,它是推断糖肽骨架的可行替代方法,特别是在肽骨架无法通过 ETD/HCD 鉴定的情况下。新的 HCD-pd-CID/ETD 工作流程将目前可用于糖蛋白质组学的最佳可能的基于决策树的 MS(2)数据采集模式结合在快速 Top Speed DDA 工作周期内。可以设想开发额外的信息学方法来挖掘和整合其中包含的丰富信息,以进行同时的 N-和 O-糖肽分析。

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