Separation of disaccharide epimers, anomers and connectivity isomers by high resolution differential ion mobility mass spectrometry

Glycans are ubiquitous, structurally diverse molecules that have specific and general roles involving metabolism, structure, energy storage, and cell-to-cell signalling. Functional specificity depends strongly on the complexity of structures that polysaccharides can adopt based on their subunit composition, length, extent of branching, glycosidic bond connectivity and anomeric configuration. However, the rapid, sensitive, and comprehensive characterization of glycan isomers can be challenging owing to limitations associated with their separation. Here, ten composition, anomeric and connectivity disaccharide isomers were separated and detected using high-resolution differential ion mobility-mass spectrometry (DMS-MS, also known as FAIMS). Focus was primarily directed to compositional isomers corresponding to epimers that differ by the axial or equatorial position of a single hydroxyl group. DMS resolving power was enhanced 15-fold primarily by increasing the fraction of helium in the ion carrier gas and lowering the flow rate. DMS-MS of each disaccharide resulted in surprisingly complex and unique multi-peak spectra with up to ten fully and partially resolved peaks for β-1,4-mannobiose (Man-1,4β-Man). Each DMS spectrum has at least one differentiating peak that is not in the other spectra, indicating that DMS can be used to fully or partially resolve composition, configuration and connectivity isomers. Thus, DMS-MS has the potential to be used for identifying polysaccharide isomers in a spectral ‘fingerprinting’ approach. The integration of high-resolution DMS into high-throughput MS-based glycomics and glycoproteomics workflows may be useful in the future for improving the characterization of glycans and glycosylated biomolecules.