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

• DOI: 10.1074/mcp.m000031-mcp201
• Publication Date: 2010-04-02T00:15:23Z
• Authors: Nichollas E. Scott; Benjamin L. Parker; Angela M. Connolly; Jana Paulech; Alistair V.G. Edw...; Ben Crossett; Linda Falconer; Daniel Kolarich; Steven P. Djordjevic; Peter Højrup; Nicolle H. Packer; Martin R. Larsen; Stuart J. Cordwell
• Keywords: Electron-transfer dissociation; Collision-induced dissociation

Campylobacter jejuni is a gastrointestinal pathogen that able to modify membrane and periplasmic proteins by the N-linked addition of 7-residue glycan at strict attachment motif (D/E)XNX(S/T). Strategies for comprehensive analysis targets glycosylation, however, are hampered resistance glycan-peptide bond enzymatic digestion or β-elimination have previously concentrated on soluble glycoproteins compatible with lectin affinity gel-based approaches. We developed strategies enriching C. HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography examined novel fragmentation, including collision-induced dissociation (CID) higher energy collisional (C-trap) (HCD) as well CID/electron transfer (ETD) mass spectrometry. CID/HCD enabled identification structure peptide backbone, allowing glycopeptide identification, whereas CID/ETD elucidation glycosylation sites maintaining linkage. A total 130 glycopeptides, representing 75 sites, were identified from LC-MS/MS coupled CID/ETD. provided majority identifications (73 sites) compared ETD (26 sites). also soybean agglutinin two-dimensional electrophoresis further six sites. This study more than doubles number confirmed in first utilize HCD fragmentation intact glycan. show hydrophobic integral significant this organism. Our data demonstrate peptide-centric approaches spectrometric techniques may be suitable application eukaryotic simultaneous structures sequence.