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Combining FAIMS based Glycoproteomics and DIA Proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in N. gonorrhoeae.
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  • Chris Hadjineophytou,
  • Edmund Loh,
  • Michael Koomey,
  • Nichollas Scott
Chris Hadjineophytou
University of Oslo
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Edmund Loh
Karolinska Institute
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Michael Koomey
University of Oslo
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Nichollas Scott
The University of Melbourne Department of Microbiology and Immunology

Corresponding Author:[email protected]

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Abstract

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of N. gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.
05 Nov 2023Submitted to PROTEOMICS
09 Nov 2023Assigned to Editor
09 Nov 2023Submission Checks Completed
09 Nov 2023Review(s) Completed, Editorial Evaluation Pending
15 Nov 2023Reviewer(s) Assigned