FAIMS enabled glycoproteomic analysis expands the known glycoproteome of N. gonorrhoeae expressing chimeric PglOs.
With the ability of FAIMS fractionation to allow the identification ofN. gonorrhoeae glycopeptides established, we sought to further probe the utility of this approach to understand glycoproteome diversity utilizing a recently reported panel of chimeric pglO alleles generated from the fusion of pglOcinerea andpglOmeningitidis expressed within N. gonorrhoeae [52]. Across this panel of 15 pglO alleles dramatic changes in glycosylation patterns are observed (Figure 2A), yet the individual glycoproteins altered, if these changes are due to changes in occupancy or glycoprotein levels, as well as how these alterations impact the broader proteome remain unclear. To assess these questions, we utilized a pooled reference approach [53] generating reference pools of each pglO allele and surveyed these glycoproteomes using FAIMS fractionation revealing dramatic differences in the number of glycopeptides and glycoproteins identified across strains (Figure 2A, Supplementary table 2 and Supplementary Data 2). Consistent with the changes observed by glycan – specific, western analysis, FAIMS based glycoproteomics supports a reduction in glycoprotein diversity within the majority of chimeric pglO alleles compared to N. gonorrhoeae expressing pglOmeningitidis (Figure 2B). At the glycopeptide level, a total of 66 unique glycopeptides were identified across all pglO alleles with the majority of FAIMS glycoproteome references only containing a subset of glycopeptides (Supplementary Figure 2). Combined, a total of 37 unique glycoproteins were identified across this panel including 26 previously uncharacterized glycoproteins (Supplementary Figure 3). Across this panel, only ~40% of glycoproteins were identified in more than 3 strains (16 glycoproteins, Figure 2C) with upset analysis revealing only five glycoproteins uniformly identified across all strains and the N. gonorrhoeae reference generated from strains expressing pglOmeningitidis possessing the highest number of unique glycoproteins (9 unique glycoproteins, Figure 2D). Importantly, FAIMS analysis recapitulates the trends in the levels of glycosylation observed by glycan specific blotting, for example, western blotting supports a marked decrease in glycosylation withinN. gonorrhoeae expressing the pglO hybrid 4 and consistent with this FAIMS analysis demonstrates only five glycoproteins were identified within this strain (Figure 2A and D). Combined, these results expand the number of substrates known to be able to be glycosylated within N. gonorrhoeae to 44 glycoproteins (Supplementary Table 4, Supplementary Figure 4) and supports that the expression of differentpglO alleles results in the alteration of the glycoproteome ofN. gonorrhoeae .