title: Enhancing structural characterisation of glucuronidated O-linked glycans using negative mode ion trap higher energy collision-induced dissociation mass spectrometry.
abstract: BACKGROUND
  High protein production and secretion with eukaryotic glycosylation machinery make T. reesei RUT-C30 a suitable expression host for recombinant proteins. The N-glycosylation of secreted proteins of RUT-C30 is known to vary depending on culture nutrients but O-glycosylation has been less extensively studied.
  
  
  METHODS
  O-Glycans and glycopeptides from secreted proteins were separated by porous graphitised carbon and C-18 liquid chromatography, respectively. O-Glycans were analysed in negative ion mode by electrospray ionisation linear ion trap mass spectrometry and glycopeptides in positive ion mode by electrospray ionisation hybrid quadrupole-orbitrap mass spectrometry. Tandem mass spectrometry was used on O-glycans and glycopeptides including ion trap higher energy collision-induced dissociation (tHCD) to detect glycan fragments not detectable with standard ion trap fragmentation. tHCD allowed targeted MS3experiments to be performed on structures containing hexuronic acid, which was not possible with ion trap CID, validating this novel O-glycan composition. Positive mode C18-LC/ESI-MS/MS was used to identify and characterise glycopeptides found to be modified with this class of O-glycans, identifying cellobiohydrolase I as a carrier of these novel O-glycans.
  
  
  RESULTS
  Negative mode ion trap higher energy collision-induced dissociation allowed detection and targeted MS3experiments to be performed on the hexuronic acid substituent of O-glycan structures, which was not possible with ion trap CID, validating the novel O-glycan composition to include hexuronic acid. Using glycopeptide analysis, this novel O-glycan composition was found to be present on the catalytic domain of cellobiohydrolase I, the most abundant secreted protein by T. reesei.
  
  
  CONCLUSIONS
  These are the first reported O-glycans to contain acidic sugars in fungi and they could have significant implications for cellobiohydrolase I structure and activity as well as the activity of recombinant proteins expressed in this host system. Copyright © 2017 John Wiley & Sons, Ltd.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.|ARC Centre of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: Christopher
authors.identifier: http://orcid.org/0000-0001-5944-6179
authors.initials: C
authors.lastname: Ashwood
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: Jodie L
authors.identifier: http://orcid.org/0000-0002-2988-7843
authors.initials: JL
authors.lastname: Abrahams
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: Helena
authors.identifier: http://orcid.org/0000-0003-0083-4630
authors.initials: H
authors.lastname: Nevalainen
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia.|ARC Centre of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW, 2109, Australia.
authors.forename: Nicolle H
authors.identifier: http://orcid.org/0000-0002-7532-4021
authors.initials: NH
authors.lastname: Packer