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

The fucosylation of glycans leads to diverse structures and is associated with many biological and disease processes. The exact determination of fucoside positions by tandem mass spectrometry (MS/MS) is complicated because rearrangements in the gas phase lead to erroneous structural assignments. Here, we demonstrate that the combined use of ion‐mobility MS and well‐defined synthetic glycan standards can prevent misinterpretation of MS/MS spectra and incorrect structural assignments of fucosylated glycans. We show that fucosyl residues do not migrate to hydroxyl groups but to acetamido moieties of N‐acetylneuraminic acid as well as 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 which can be used to accurately determine fucosyl positions in glycans.