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

Abstract

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.

Fucose Migration Pathways Identified Using Infrared Spectroscopy**

“…Overall, our findings indicate that fucose is prone to migrate towards the O6 position of GlcNAc in the gas phase, regardless of its initial location. Because fucose does not migrates towards hexoses, it was suggested that it rearranges to the oxygen or nitrogen of the acetamido moiety [17] . Our findings indicate that the fucose migrates towards hydroxyl groups instead.…”

“…To date, as a result of the proposed in‐source migration mechanism, individual fucosylated structures could not be isolated for further IR analysis. This observation was confirmed by IM‐MS [17] . In this context, structural data supporting that the structure of protonated fucosylated structures could be retained at all through MS workflows, as well as reference data supporting the structural characterization of fucosylated products were lacking.…”

“…using MS/IR and by Boons et al. using IM‐MS: fucose might migrate even in an intact glycan, without the need for CID [16, 17] . Fucose migration was previously considered to be triggered by CID, these works suggest that migration could also occur during the ionization and/or the desolvation process or during transfer of the ions through the mass spectrometer.…”

Fucose Migration Pathways Identified Using Infrared Spectroscopy**

“…Recent works by Mucha et al [16] . and Toraño et al [17] . showed that migration of fucose from one residue to another may occur as early as at the ionisation stage in a mass spectrometric workflow using IR spectroscopy and ion mobility.…”

State‐of‐the‐art glycosaminoglycan characterization

“…For example, it was shown that synthetic oligosaccharide isomers can be successfully separated using TWIMS, despite only minor differences in their regio‐ and stereo‐chemistry at a singly glycosidic bond (Hofmann et al, 2015). Furthermore, it was demonstrated that fragment‐based approaches can identify fucosylated (Sastre Toraño et al, 2019) and sialylated linkages (Hofmann & Pagel, 2017; Hofmann et al, 2017; Lane et al, 2019) which can be used to determine characteristic features on milk oligosaccharides and complex N ‐glycans (Harvey et al, 2018b; Pagel & Harvey, 2013). The combination of mass measurement and IM‐MS analysis also enabled the assignment and identification of isomeric glycopeptides and separation into different charge states (Creese & Cooper, 2012; Zhu et al, 2015).…”