Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID, Higher Energy Collisional Dissociation, and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni
Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID, Higher Energy Collisional Dissociation, and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni
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DOI：
10.1074/mcp.m000031-mcp201
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发表时间：
2011-02-01
影响因子：
7
通讯作者：
Cordwell, Stuart J.
中科院分区：
文献类型：
--
作者：
Scott, Nichollas E.;Parker, Benjamin L.;Cordwell, Stuart J.
空肠弯曲杆菌是一种胃肠道病原体，其能够通过在严格附着基序（D/E）XNX（S/T）处N-连接添加7-残基聚糖来修饰膜和周质蛋白。然而，糖基化目标的全面分析策略受到聚糖-肽键对酶消化或β-消除的抗性的阻碍，并且先前集中在与凝集素亲和力和基于凝胶的方法相容的可溶性糖蛋白上。我们开发了富集C的策略。使用两性离子亲水相互作用色谱法对空肠HB 93 -13糖肽进行了分析，并检查了新的碎片化，包括碰撞诱导解离（CID）和高能碰撞（C-trap）解离（HCD）以及CID/电子转移解离（ETD）质谱法。CID/HCD能够鉴定聚糖结构和肽骨架，从而能够鉴定糖肽，而CID/ETD能够通过维持聚糖-肽连接来阐明糖基化位点。使用偶联CID/HCD和CID/ETD的两性离子亲水相互作用色谱法，从LC-MS/MS中鉴定了总共130个糖肽，代表75个糖基化位点。CID/HCD提供了大多数鉴定（73个位点），而ETD（26个位点）。我们还研究了可溶性糖蛋白的大豆凝集素亲和和双向电泳，并确定了另外六个糖基化位点。这项研究使C中确认的N-连接糖基化位点的数量增加了一倍多。jejuni，并且是第一个利用HCD片段化以完整聚糖鉴定糖肽的人。我们还表明，疏水性的膜蛋白是重要的糖基化在这种生物体的目标。我们的数据表明，肽为中心的方法耦合到新的质谱裂解技术可能适用于应用到真核糖蛋白的聚糖结构和肽序列的同时阐明。Molecular & Cellular Proteomics 10：10.1074/mcp. M000031-MCP 201，1-18，2011.
Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E) XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or beta-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation ( CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence. Molecular & Cellular Proteomics 10:10.1074/mcp.M000031-MCP201, 1-18, 2011.