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.