Glycan-tailored glycoproteomic analysis reveals Serine is the sole residue subjected to O-linked glycosylation in Acinetobacter baumannii

ABSTRACT: Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii, O-linked glycosylation is required for virulence however the targets and conservation of glycosylation events remains poorly defined. Within this work we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan Electron-Transfer/Higher-Energy Collision Dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD triggering regimes to complementary glycopeptide enrichment approaches we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (Titanium dioxide) and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (Trypsin, GluC, Pepsin and Thermolysis) we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. In contrast to earlier reports that suggested glycosylation occurred on both Threonine and Serine residues we demonstrate that Serine is the sole residue subjected to glycosylation with the substitution of Threonine for Serine abolishing glycosylation in model glycoproteins. Consistent with the requirement of Serine for glycosylation, examination of the A. baumannii pan-genome (n=567) supports that both glycoproteins and the Serine residues known to be subjected to glycosylation are highly conserved across A. baumannii isolates. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O-linked glycosylation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Acinetobacter Baumannii Atcc 19606 = Cip 70.34 = Jcm 6841

SUBMITTER: Nichollas Scott  

LAB HEAD: Nichollas E.

PROVIDER: PXD050066 | Pride | 2024-07-12

REPOSITORIES: Pride