Protein glycosylation is a ubiquitous process observed across all domains of life. Recently glycoproteomic enrichment approaches and informatic tools have seen dramatic improvements in performance, greatly expanding the accessibility of glycosylation analysis. However, for typical forms of glycosylation, such as bacterial glycosylation, the utility of these improvements remains to be systemically assessed. This is especially true for bacterial protein glycosylation systems with highly variable glycan compositions, such as the nosocomial pathogen Acinetobacter baumannii. Within Acinetobacter baumannii the O-linked substrates of protein glycosylation are poorly understood yet glycosylation is known to play essential roles within biofilm formation and virulence. To understand the function of glycosylation within this pathogen, defining the optimal approaches for deep glycoproteomic analysis of isolates with diverse O-linked glycans is essential.
Within this work we evaluate glycoproteomics approaches for the characterisation of three A. baumannii strains possessing diverse glycan structures ATCC19606, BAL062, and D1279779. We demonstrate the value of incorporating glycan-specific diagnostic ions to enhance data collection of bacterial glycoforms. Using tailored glycan-specific diagnostic ion collection methods we assessed, various 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, to expand the coverage of the A. baumannii glycoproteome revealing a total of 33 glycoproteins. This work dramatically improves our understanding of A. baumannii glycosylation localising 40 glycosylation events, of which 29 are previously unreported. Bioinformatic analysis reveals both glycoproteins and sites of glycosylation are highly conserved across A. baumanni isolates. Across identified glycosylation sites we find only serine is subjected to glycosylation with molecular approaches confirming this requirement within two native glycoproteins (ABD1_29350 and ABD1_01410). Combined, this research significantly expands our current knowledge of the A. baumannii glycoproteome at a site specificity resolution with the conservation of glycosylation sites across A. baumannii isolates indicating a strong evolutionary pressure to maintain these events.