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  • 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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    Author(s)
    Scott, Nichollas E
    Parker, Benjamin L
    Connolly, Angela M
    Paulech, Jana
    Edwards, Alistair VG
    Crossett, Ben
    Falconer, Linda
    Kolarich, Daniel
    Djordjevic, Steven P
    Hojrup, Peter
    Packer, Nicolle H
    Larsen, Martin R
    Cordwell, Stuart J
    Griffith University Author(s)
    Kolarich, Daniel
    Packer, Nicki
    Year published
    2011
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    Abstract
    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 β-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 ...
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    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 β-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.
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    Journal Title
    Molecular and Cellular Proteomics
    Volume
    10
    Issue
    2
    DOI
    https://doi.org/10.1074/mcp.M000031-MCP201
    Copyright Statement
    This research was originally published in Molecular & Cellular Proteomics (MCP). Scott et al, 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, Molecular & Cellular Proteomics (MCP), 2011; 10(2): M000031-MCP201. Copyright the American Society for Biochemistry and Molecular Biology. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitve version.
    Subject
    Biochemistry and cell biology not elsewhere classified
    Publication URI
    http://hdl.handle.net/10072/336663
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    • Journal articles

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