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Abstract

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 beta-1,4- and beta-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 beta-1,4- and beta-1,3-linked carbohydrates can be distinguished
based on diagnostic 3,5A fragment ions that are characteristic for
beta-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 beta-1,4-
and beta-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.