Discussion
Within this work, we demonstrate the utility of FAIMS based
fractionation and DIA analysis for probing bacterial glycosylation
systems revealing glycosylation changes observed in response to chimericpglO alleles in N. gonorrhoeae occur largely independent
of changes in glycoprotein levels. Benchmarking the use of FAIMS
fractionation for N. gonorrhoeae we show the glycoproteome ofN. gonorrhoeae can effectively be studied without the need for
bespoke affinity reagents and that novel glycopeptides can be readily
identified using this approach. Importantly this work shows that despite
the absence of protein abundance changes in glycoproteins alterations
within the broader proteome of N. gonorrhoeae are readily
identifiable in response to changes in glycosylation patterns.This work builds on our previous exploration of PglO targeting
specificities [52] and supports that
PglO protein substrate recognition is discrete yet reprogrammable by the
manipulation of PglO. While early studies have noted differences in the
compatibility of O- oligosaccharyltransferases to target
substrates [74], our observations
here expand on these findings demonstrating this also occurs outside of
heterologous expression systems. Our work here supports that differences
in PglO targeting ranges may be more common than previously thought
which has important ramifications for predicting the targets of
bacterial glycosylation across genera as well as unlocking the potential
of these enzymes for glycoengineering
[75].
While the use of FAIMS fractionation has been noted to be advantageous
for glycoproteomics typically improving glycoprotein/glycopeptide
coverage by as much as 75% [35,
38], within this study we observed a
>200% increase in the number of identified glycoproteins
in N. gonorrhoeae compared to earlier studies
[26,
48]. While it is likely that this
increase is due to multiple factors including the use of current
generation MS instrumentation, as well as the glycosylation of proteins
not natively glycosylated within N. gonorrhoeae by chimericpglO alleles, it is noteworthy that many novel glycoproteins
identified here correspond to integral membrane proteins containing
multiple transmembrane domains (Supplementary table 4). Previous studies
exploring the N. gonorrhoeae glycoproteome have traditionally
used protein centric approaches including protein level antibody-based
enrichment [26] or 2D gel
electrophoresis coupled with antibody-based detection
[48] which while effective are known
to be incompatible with poorly soluble hydrophobic membrane proteins
[76,
77]. The use of peptide-centric
analysis, as undertaken here, is known to enhance both the detection as
well as the characterization of modifications within bacterial membrane
glycoproteins [24]. While most
previously known N. gonorrhoeae glycoproteins were identified
using FAIMS fractionation, glycopeptides derived from four previously
identified glycoproteins failed to be detected with two of these
proteins (NGO_1237 and NGO_0994) detected by DIA analysis and the
remaining two glycoproteins either not detected (NGO_0983) or absent
due to being disrupted within these backgrounds (PilE, NGO_1177).
Importantly, while NGO_1237 and NGO_0994 are both known to be
glycosylated the precise location of glycosylation within NGO_0994 is
yet to be confirmed being predicted to be located in a region lacking
Lysines / Arginines [52] and
glycosylation within NGO_1237 requiring alternative enzymes to Trypsin
to be accessible [48,
78]. Combined, these results support
that for the Trypsin accessible glycoproteome, FAIMS based analysis
provides broad coverage of the known glycoproteome and provides access
to novel membrane glycoproteins.
The identification that changes in glycosylation patterns impact theN. gonorrhoeae proteome contributes to a growing body of work
linking proteome alterations with changes in glycosylation occupancy
across a range of Gram-negative species
[79-81]. Interestingly while previous
studies have explored the impact of the abolishment of glycosylation few
studies have assessed how changes in glycosylation occupancy or
glycosylation patterns impact bacterial proteomes. Recently it was
demonstrated that within Burkholderia species the silencing of
glycosylation using CRISPRi reduced occupancy but resulted in only
modest proteomic impacts [82] yet in
contrast our findings here support the N. gonorrhoeae proteome is
extensively impacted by changes in glycosylation patterns. WhileO- linked glycosylation within B. cenocepacia has been
shown to be important for the stability of multiple proteins
[81,
83] we observed no change in the
protein abundance of glycoproteins within this work suggesting that
within N. gonorrhoeae O- linked glycosylation may exert its
effects by modulating protein interactions or protein activities.
Consistent with this previous work has shown for N. gonorrhoeaePilE glycosylation is not required for Pilin formation yet appears to
enhance the stability of this structure
[84] and for other forms of bacterial
glycosylation, such as N-linked glycosylation within C. jejuni,glycosylation has also been shown to stabilise protein complexes with
this effect occurring independent of changes in protein abundance
[85]. Thus, while we find the
abundance of glycoproteins are unaffected future work may seek to
confirm if the glycosylation events identified here also play roles in
modulating protein functions and/or the stabilization of protein
complexes driving the proteomic effects observed here.
Within this study, our implementation of DIA analysis allowed access to
proteome as well as glycosylation occupancy information yet, it is
important to note that many glycopeptides observed by FAIMS
fractionation are not observable within our DIA dataset due to the
imperfect overlap in the peptides observed between these two approaches.
As previously noted, FAIMS fractionation preferentially enables access
to large (>1000 m/z), low charge density glycopeptides
[35] with a similar trend also
observed for N. gonorrhoeae (Supplementary Figure 1B). Due to
this bias, most N. gonorrhoeae glycopeptides fall outside the m/z
range utilised in our DIA analysis (350 to 951 m/z) which has been
tailored to maximize the proteome coverage
[53]. While DIA methods compatible
with glycopeptides are emerging [86,
87], these methods consciously utilize
wider DIA m/z ranges which improve access to glycopeptides yet this can
lead to more congested MS/MS spectra reducing DIA depth
[53]. While enzymatic
[88,
89] and genetic
[90] approaches can be used to reduce
glycan sizes and heterogeneity to enhance the detection of both
glycosylated and non-glycosylated forms of peptides the size of the
native N. gonorrhoeae trisaccharide (552Da) limits the
feasibility of using identical analysis approaches. Despite this caveat
we find the non-glycosylated forms of 50 peptides subjected to
glycosylation could be observed using DIA supporting that while not
optimized to allow coverage of both the glycoproteome and proteome
information on glycosylation occupancy is still readily accessible.
Interestingly within this analysis, we observed the changes in the
non-glycosylated forms of peptides is generally modest, less than a
<40% reduction in intensity with only a few examples of
peptides unable to be identified between different strains (Figure 5 and
Supplementary Figure 7 to 10). Thus, this relatively small impact on
abundance suggests that the overall glycosylation occupancy may be
modest for many N. gonorrhoeae glycoproteins observed here.
In summary, this work furthers our understanding of the utility of FAIMS
based fractionation for bacterial glycoproteomics and expanse the known
glycoproteome of N. gonorrhoeae . This work contributes to a
growing body of studies that demonstrates that bacterial
oligosaccharyltransferases possess discrete targeting ranges and that
the manipulation of these enzymes can vary the observed glycosylation
pattern within bacterial systems. Our identification that changes inO -linked glycosylation patterns leads to changes in the proteome
further supports that glycosylation plays multiple roles in N.
gonorrhoeae biology. Finally, this work demonstrates how glycoproteomic
insights and peptide centric DIA information can be integrated to
provide support for changes in occupancy across N. gonorrhoeaestrains expressing different pglO chimeras. These changes in
glycosylation patterns largely appear independent of changes in
glycoprotein levels and peptide analysis supports that glycosylation
occupancy is generally low within most observed glycoproteins.