Oral Presentation 26th Annual Lorne Proteomics Symposium 2021

Characterisation of the O-glycoproteome of Porphyromonas gingivalis (#31)

Paul D Veith 1 , Nichollas E Scott 2 , Eric C Reynolds 1
  1. Melbourne Dental School, Bio21 Institute, University of Melbourne, Parkville, Victoria, Australia
  2. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia

Porphyromonas gingivalis is a Gram-negative oral pathogen belonging to the Bacteroidetes phylum. A general O-glycosylation system targeting exported proteins has been reported in this phylum and the major O-glycans have been characterized in a few species. In this study we identify the major O-glycans in P. gingivalis and identify the glycosylation sites. Proteins from membrane fractions were either fractionated by SDS-PAGE or subjected to glycoprotein enrichment using ZIC-HILIC. Proteins were digested with trypsin and analysed by LC-MS/MS with an Orbitrap equipped with a FAIMS source. Two different FAIMS settings were used and peptides containing the HexNAc oxonium ion were selected for ETD and CID fragmentation to aid elucidation of the glycosylation site and glycan sequence respectively. In a complementary approach, protein fractions were also partially deglycosylated with trifluoromethanesulfonic acid (TFMS) prior to MS analyses. So far, ~320 unique peptide sequences encompassing ~200 sites from ~120 proteins have been identified. The proteins included surface proteins, outer membrane proteins, lipoproteins and periplasmic proteins. The proteins exhibited between one and seven glycosylation sites which conformed to the known motif of D[T/S]X, where X was known to include A,V,L,I, M and T. In P. gingivalis we now show that X can also be S, C, F and G. The two major glycans have delta masses of 1436 and 1394 respectively and appear to be the same apart from an additional acetyl group. The preliminary glycan sequence of the 1436 Da form is Hex-(HexA)-Hex-(dHex)-dHex-HexNAc-X-HexNAc where X=238 Da. With the use of deglycosylation, the advantage of being able to select for HexNAc-containing glycopeptides was lost, however it was beneficial for identifying additional glycopeptides and was very helpful for glycan sequencing since it truncated the glycans to form a ladder. In addition, the truncated glycoforms yielded ETD fragmentation patterns more amenable to identification of glycosylation sites.