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 .