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In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways.

A cationic cysteine-hydrazide as an enrichment tool for the mass spectrometric characterization of bacterial free oligosaccharides