New insights into the type A glycan modification of Clostridioides difficile flagellar protein flagellin C by phosphoproteomics analysis |
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Authors: | Paul J Hensbergen Arnoud H de Ru Annemieke H Friggen Jeroen Corver Wiep Klaas Smits Peter A van Veelen |
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Institution: | 1.Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands;2.Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands |
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Abstract: | The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility. The study of post-translational modifications often relies on some type of enrichment strategy; however, a procedure for enrichment of this modification has not yet been demonstrated. In this study, we show that an approach that is commonly used in phosphoproteomics, Fe3+-immobilized metal affinity chromatography, also enriches for peptides with this unique post-translational modification. Using LC–MS/MS analyses of immobilized metal affinity chromatography–captured tryptic peptides, we observed not only type A-modified C. difficile flagellin peptides but also a variety of truncated/modified type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a type A structure containing a phosphonate (2-aminoethylphosphonate), a modification that is rarely observed and has hitherto not been described in C. difficile. In conclusion, we show that a common enrichment strategy results in reliable identification of peptides carrying a type A glycan modification, and that the results obtained can be used to advance models about its biosynthesis. |
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Keywords: | phosphonate motility Clostridioides mass spectrometry IMAC glycosylation 2-aminoethylphosphonate |
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