Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Identification of novel N-glycosylation sites from the bacterium Campylobacter jejuni (#448)

Joel Cain 1 2 , Stuart Cordwell 1 2 3 4
  1. School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia
  2. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia
  3. Mass Spectrometry Core Facility , University of Sydney, Sydney, NSW, Australia
  4. Discipline of Pathology, University of Sydney, Sydney, NSW, Australia


One of the prominent causative agents of acute gastroenteritis in the developed world is Campylobacter jejuni. An important factor for virulency, as well as its ability to establish a commensal relationship within avian guts (the primary vector for infection), is its capacity to perform protein N-linked glycosylation. Recently we established that loss of N-glycosylation was associated with an array of phenotypes including aberrant chemotactic responses, altered respiratory preferences, changes in cell morphology and increased susceptibility towards temperature and osmotic stress. Given the lack of concrete connections between known targets of this PTM and the affected phenotypic traits, we looked to broaden the list of known N-glycoproteins within C. jejuni with the aim of identifying putative causative agents.


Membrane proteins from C. jejuni 11168 O were enriched using Na2CO3 precipitation, following protein aliquots were digested using an array of commercially available proteolytic enzymes. Intact N-glycopeptides were then enriched using ZIC-HILIC. N-glycopeptides were analysed on an Orbitrap Fusion™ Tribrid™ mass spectrometer using HCD and product-ion triggered CID fragmentation.

We also looked to identify N-glycoproteins from 11168 O ΔpglB::pglB with the rationale that the reported elevated abundance of the oligosaccharyltransferase, PglB, within this strain would lead to elevated levels of low abundance N-glycopeptides. 

Key findings:

Of the known 131 modification sites, we were able to unequivocally demonstrate occupation of ~80% of known N-glycopeptides. We were able to identify 54 novel modification sites, including 15 novel N-glycoproteins.