Ion‐Mobility Spectrometry Can Assign Exact Fucosyl Positions in Glycans and Prevent Misinterpretation of Mass‐Spectrometry Data After Gas‐Phase Rearrangement

Abstract

Fucosylation of glycans is structurally diverse and has been associated with many biological and disease processes. Exact determination of fucoside positions by tandem mass spectrometry (MS/MS) is complicated by rearrangements in the gas‐phase leading to erroneous structural assignments. Here, we demonstrate that the combined use of ion mobility (IMS)‐MS and well‐defined synthetic glycan standards can prevent misinterpretation of MS/MS spectra and avoid incorrect structural assignments of fucosylated glycans. We show that fucosyl residues do not migrate to hydroxyls but to acetamido moieties of N‐acetylneuraminic acid and N‐acetylglucosamine residues and nucleophilic sites of an anomeric tag, yielding specific isomeric fragment ions. This mechanistic insight enables the characterization of unique IMS arrival time distributions of the isomers, that can be used to accurately determine fucosyl positions in glycans. These results, in turn, facilitate unambiguous discrimination between MS/MS fragments arising from parent compounds and those that occur due to rearranged fucosyl residues preventing misinterpretation of MS/MS spectra.