Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

Abstract

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species

“…In order to confirm this, we analyzed selected glycosides by ion-mobility MS (IM-MS), a rapid and sensitive technique that separates gas phase ions based on their mobility (inversely proportional to a molecules collision cross-section area) as they traverse through an inert buffer gas [33, for review see 34]. IM-MS has been previously reported as being able to discern the identity of hexose residues based on the arrival time distribution (ATD) of monosaccharide product ions, generated by collision-induced dissociation (CID) of either glycopeptides or free reducing-saccharides [17]. Recently it has also been shown that IM-MS has the potential to discern the stereo-/regio-chemistry of the glycosidic bond of glucose oligosaccharides, based on the ATDs of these monosaccharide product ions compared to synthetic standards (C.J.…”

“…Samples were analysed according to the protocol reported by Both et al [17]. Briefly, plant glycosides were diluted to approx.…”

“…The temporal as well as spatial occurrence of these turnover intermediates is investigated, and their function in plant physiological processes proposed. We also highlight the application of a new method involving the use of ionmobility mass spectrometry (IM-MS) as an analytical tool capable of sequencing the monosaccharide building blocks within the glycosides studied [17].…”

Complete Hexose Isomer Identification with Mass Spectrometry

“…Interestingly, in a study of the analysis of epimeric glycans with ion-mobility mass spectrometry, the presence of a single axial hydroxyl substituent on an aldohexose monosaccharide was hypothesized to lead to a lack of discrimination, as in the case of galactose (C4) versus mannose (C2) [6]. This single axial hydroxyl theory, however, does not hold in this fixed ligand method described.…”

“…However, carbohydrate sequencing has significantly lagged capabilities in sequencing nucleic acids and proteins. Compared with these other biomolecules, polymers and oligomers of carbohydrates are more structurally complex because of their large range of stereochemical differences, absolute configuration (D or L), anomeric configurations (α or β), and linkage positions [2,4,6,7]. This high degree of structural complexity has hindered accurate structural analyses of glycans, so much so that a United States National Academy of Sciences report issued in 2012 has called for the development of technology over the next 10 years to purify, identify, and determine the structures of all the important glycoproteins, glycolipids, and polysaccharides in any biological sample [8].…”