FAIMS fractionation allows monitoring of the N.
gonorrhoeae glycoproteome.
The identification of N. gonorrhoeae glycoproteins has
traditionally relied on the detection and/or enrichment of glycoproteins
using bespoke antibodies which, while effective
[26,
48], has restricted the
characterization of the N. gonorrhoeae glycoproteomes to
specialized laboratories. Currently at least 19 proteins are known to be
glycosylated in N. gonorrhoeae[26] yet the full extent of protein
glycosylation within this species is unclear. Utilizing FAIMS
instrumentation, we previously demonstrated bacterial glycopeptides
could be isolated from complex lysates providing a complementary
technique to affinity enrichment approaches for assessing bacterial
glycoproteomes [35]. Thus, to improve
our understanding of the N. gonorrhoeae glycoproteome and to
further benchmark the abilities of FAIMS to isolate bacterial
glycopeptides, we assessed the identification of N. gonorrhoeaeMS11 glycopeptides across differing FAIMS CVs from -20 to -80 (Figure
1A-D, Supplementary Table 1 & Supplementary Data 1). Consistent with
our previous studies [35], we observe
that FAIMS fractionation readily allows the identification of
glycopeptides across different FAIMS CVs (Figure 1A) with the majority
of glycopeptides identified within CVs above -50 and a CV of -30
enabling access to the highest number of unique glycopeptides across
replicates (Figure 1A-C). Across three biological replicates, a total of
48 unique glycopeptides were identified with >70% of
identified glycopeptides (34 glycopeptides) observed across all
replicates (Supplementary figure 1A). At the protein level, a total of
26 unique glycoproteins were identified with 13 corresponding to
previously identified glycoproteins
[26] (Figure 1D). Interestingly,
across the 48 unique glycopeptides only 7 glycosylation sites were
definitively localized reflecting the low charge density of these
glycopeptides and supporting previous studies demonstrating the
preference for serine glycosylation by PglO (Supplementary Figure 1B
&C, Supplementary Data 1). Taken together, these results support FAIMS
fractionation enables glycoproteomic analysis of N. gonorrhoeaeto a comparable depth to previous approaches and allows the
identification of novel glycoproteins.