Oral Presentation 26th Annual Lorne Proteomics Symposium 2021

Microbial glycoproteomics; how do you find glycopeptides when you’re not sure what you’re looking for? (#30)

Nichollas E Scott 1
  1. Department of Microbiology and Immunology, University of Melbourne, Melbourne, VICTORIA, Australia

Microbial glycoproteomics is a rapidly growing field which seeks to characterise glycosylation at a proteome scale in Bacteria and Protozoa. Although interest in microbial glycoproteins as potential biomarkers or vaccine candidates has increased the atypical nature of these events has limited the study of microbial glycosylation to a hand full of specialised laboratories. To improve the accessibility of microbial glycosylation we have sought to develop agnostic bioinformatic and enrichment approaches to streamline the analysis of microbial glycoproteomes using members of the Burkholderia genus as model systems. Using open searching we have now shown that microbial glycosylation events can be identified in an unsupervised manner without prior knowledge of the glycans used for glycosylation. Using open searching determined glycan profiles we have also shown that glycan utilisation can be rapidly compared allowing genus wide comparisons of glycosylation diversity. To streamline the identification of microbial glycopeptides we have also explored the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation demonstrating that at high compensation voltages (CVs) short aliphatic glycopeptides can be readily isolated. This FAIMS based glycopeptide enrichment enables glycopeptide selection from complex samples providing an alternative means to identify glycopeptides recalcitrant to hydrophilic based enrichment approaches. Combining these approaches, we have now shown the glycoproteome of Burkholderia species are nearly 10 times larger than initially thought (~200 glycoproteins per species) and that O-linked glycosylation only occurs on serine residues. Furthermore, by combining multiple FAIMS CVs within a single analytical run this enables both proteome and glycoproteome analysis of even limited samples. Taken together, these results demonstrate that open searching and FAIMS enrichment approaches are valuable tools for glycoproteomic analysis.