DIA analysis supports alterations in glycosylation occupancy between pglO alleles.
The observation that across the N. gonorrhoeae proteome only a single glycoprotein is impacted by the expression of differentpglO alleles supports alterations in glycosylation reflect changes in occupancy rather than changes in glycoprotein levels. Across the 44 glycoproteins identified within N. gonorrhoeae using DIA analysis, we observed 40 of these glycoproteins are quantified with > 10 unique precursors highlighting the ability of DIA analysis to provide high protein sequence coverage of the N. gonorrhoeae glycoproteome (Supplementary Figure 6). Due to this high coverage, we sought to directly gauge the relative levels of glycosylation using the non-glycosylated forms of glycopeptides inspired by recent studies highlighting the ability of PTM status and isoform specific information to be retrievable using peptide-centric analysis [71-73]. Within our DIA dataset, 59.5% (50 out of 84) peptide sequences observed glycosylated within FAIMS experiments (Supplementary table 1 and 2) were identifiable in their non-glycosylated forms with 39.3% (33 out of 84) corresponding to fully cleaved peptide sequences suitable for quantification assessments (Figure 5A, Supplementary Table 6). Overlaying protein and peptide quantification information in combination with information on the glycosylation status as determined from FAIMS analysis, we observe that while the protein levels of glycoproteins remain constant acrossN. gonorrhoeae strains expressing different pglO alleles, peptides observed to undergo glycosylation display alterations across biological groups consistent with changes in occupancy (Figure 5B-D, Supplementary Figure 7-10). For example, within the glycoprotein NGO_1365, the peptide384EWAPSENQAAAPQAGVQTASEAKPASEAK412shows consistent and similar measurements within biological groups but across groups notable variations within the peptide abundance are observed (Figure 5B). Consistent with changes in glycosylation, we also observed the absence of unglycosylated peptides within some biological groups suggesting potential enhanced occupancy of these peptides with specific pglO alleles. For example, within the glycoproteins NGO_1800, the peptide173VAVGVQTASGAQTVR187 was observed across all biological groups but is absent in 3 out of the 4 biologicals of pglOmeningitidis expressed within N. gonorrhoeae (Figure 5C). Similarly, for the glycoprotein NGO_2092 we observe the peptide31EQAVSAAQSESASVTVK50 can be consistently identified within biological groups corresponding topglO hybrid 1, 2, 4, 5, 6, 8, 9 and 11 but is absent in all other groups with glycosylation being observed on this peptide inpglOcinerea and pglO hybrid 3, hybrid 7, 9, 10 and 12 (Figure 5D). These results support that the alterations observed within the N. gonorrhoeae glycoproteome by western and FAIMS analysis correspond to changes in occupancy with the intensity / the detectability of multiple non-glycosylated forms of known glycopeptide altered across strains expressing different pglOalleles.