The connection between monosaccharides influences the structure, solubility, and biological function of carbohydrates. Although tandem mass spectrometry (MS/MS) often enables the compositional identification of carbohydrates, traditional MS/MS fragmentation methods fail to generate abundant cross-ring fragments of intrachain monosaccharides that could reveal carbohydrate connectivity. We examined the potential of helium-charge transfer dissociation (He-CTD) as a method of MS/MS to decipher the connectivity of β-1,4- and β-1,3-linked oligosaccharides. In contrast to collision-induced dissociation (CID), He-CTD of isolated oligosaccharide precursors produced both glycosidic and cross-ring cleavages of each monosaccharide. The radical-driven dissociation in He-CTD induced single cleavage events, without consecutive fragmentations, which facilitated structural interpretation. He-CTD of various standards up to a degree of polymerization of 7 showed that β-1,4- and β-1,3-linked carbohydrates can be distinguished based on diagnostic 3,5A fragment ions that are characteristic for β-1,4-linkages. Overall, fragment ion spectra from He-CTD contained sufficient information to infer the connectivity specifically for each glycosidic bond. When testing He-CTD to resolve the order of β-1,4- and β-1,3-linkages in mixed-linked oligosaccharide standards, He-CTD spectra sometimes provided less confident assignment of connectivity. Ion mobility spectrometry-mass spectrometry (IMS-MS) of the standards indicated that ambiguity in the He-CTD spectra was caused by isobaric impurities in the mixed-linked oligosaccharides. Radical-driven dissociation induced by He-CTD can thus expand MS/MS to carbohydrate linkage analysis, as demonstrated by the comprehensive fragment ion spectra on native oligosaccharides. The determination of connectivity in true unknowns would benefit from the separation of isobaric precursors, through UPLC or IMS, before linkage determination via He-CTD.
Keywords: branching pattern determination; ion/ion activation; isomers; oligosaccharides; structural characterization.