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

Scott, NE; Parker, BL; Connolly, AM; Paulech, J; Edwards, AVG; Crossett, B; Falconer, L; Kolarich, D; Djordjevic, SP; Højrup, P; Packer, NH; Larsen, MR; Cordwell, SJ

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

Scott, NE Parker, BL Connolly, AM Paulech, J Edwards, AVG Crossett, B Falconer, L Kolarich, D Djordjevic, SP

Højrup, P Packer, NH Larsen, MR Cordwell, SJ

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Publication Type:: Journal Article
Citation:: Molecular and Cellular Proteomics, 2011, 10 (2)
Issue Date:: 2011-02-01

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Field	Value	Language
dc.contributor.author	Scott, NE	en_US
dc.contributor.author	Parker, BL	en_US
dc.contributor.author	Connolly, AM	en_US
dc.contributor.author	Paulech, J	en_US
dc.contributor.author	Edwards, AVG	en_US
dc.contributor.author	Crossett, B	en_US
dc.contributor.author	Falconer, L	en_US
dc.contributor.author	Kolarich, D	en_US
dc.contributor.author	Djordjevic, SP https://orcid.org/0000-0001-9301-5372	en_US
dc.contributor.author	Højrup, P	en_US
dc.contributor.author	Packer, NH	en_US
dc.contributor.author	Larsen, MR	en_US
dc.contributor.author	Cordwell, SJ	en_US
dc.date.issued	2011-02-01	en_US
dc.identifier.citation	Molecular and Cellular Proteomics, 2011, 10 (2)	en_US
dc.identifier.issn	1535-9476	en_US
dc.identifier.uri	http://hdl.handle.net/10453/18680
dc.description.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 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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.	en_US
dc.relation.ispartof	Molecular and Cellular Proteomics	en_US
dc.relation.isbasedon	10.1074/mcp.M000031-MCP201	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.subject.mesh	Cell Membrane	en_US
dc.subject.mesh	Campylobacter jejuni	en_US
dc.subject.mesh	Soybeans	en_US
dc.subject.mesh	Glycopeptides	en_US
dc.subject.mesh	Polysaccharides	en_US
dc.subject.mesh	Peptides	en_US
dc.subject.mesh	Chromatography	en_US
dc.subject.mesh	Electrophoresis, Gel, Two-Dimensional	en_US
dc.subject.mesh	Protein Structure, Tertiary	en_US
dc.subject.mesh	Electron Transport	en_US
dc.subject.mesh	Glycosylation	en_US
dc.subject.mesh	Genome	en_US
dc.subject.mesh	Electrons	en_US
dc.subject.mesh	Mass Spectrometry	en_US
dc.subject.mesh	Hydrophobic and Hydrophilic Interactions	en_US
dc.title	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	en_US
dc.type	Journal Article
utslib.citation.volume	2	en_US
utslib.citation.volume	10	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	0605 Microbiology	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	closed_access
pubs.issue	2	en_US
pubs.publication-status	Published	en_US
pubs.volume	10	en_US

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 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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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http://hdl.handle.net/10453/18680