Abstract
There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewisx and Lewisy epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone.Graphical abstractᅟ
Highlights
A pproximately half of all proteins are estimated to be glycosylated either at asparagine in an Asn-Xxx-Ser(Thr) motif where Xxx is any amino acid except proline or to serine or threonine
Negative ion collision-induced dissociation (CID) alleviates the isomer problem when accompanied by ion mobility and this latter technique has proved to be invaluable for isolating glycan-derived ions from contaminated mixtures, minimizing extensive pre-mass spectrometric cleanup strategies with their accompanying sample losses [25, 26]
We have developed these techniques for the analysis of high-mannose [14, 15, 17, 36], hybrid [16, 17, 37], and complex N-glycans [16, 17, 19, 20, 25, 37,38,39,40,41] but little attention has so far been paid to glycans with multiple fucose residues present on the core GlcNAc and on their antennae
Summary
A pproximately half of all proteins are estimated to be glycosylated either at asparagine in an Asn-Xxx-Ser(Thr) motif where Xxx is any amino acid except proline (termed Nlinked) or to serine or threonine (termed O-linked). These glycans are involved in many processes, such as protein folding, cell-cell recognition, and protein turnover [1], and methods for their structural determination have been developed by many. Negative ion CID alleviates the isomer problem when accompanied by ion mobility and this latter technique has proved to be invaluable for isolating glycan-derived ions from contaminated mixtures, minimizing extensive pre-mass spectrometric cleanup strategies with their accompanying sample losses [25, 26]. Ion mobility combined with negative ion CID provides an excellent method for N-glycan analysis [27,28,29,30,31,32,33,34,35]
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