Identification of N-glycan positional isomers by combining IMS and vibrational fingerprinting of structurally determinant CID fragments

While glycans are present on the surface of cells in all living organisms and play key roles in most biological processes, their isomeric complexity makes their structural characterization challenging. Of particular importance are positional isomers, for which analytical standards are difficult to obtain. We combine ultrahigh-resolution ion-mobility spectrometry with collision-induced dissociation and cryogenic infrared spectroscopy to determine the structure of N-glycan positional isomers. This approach is based on first separating the parent molecules by SLIM-based IMS, producing diagnostic fragments specific to each positional isomer, separating the fragments by IMS, and identifying them by comparing their IR fingerprints to a previously recorded spectral database. We demonstrate this strategy using a bottom-up scheme to identify the positional isomers of the N-linked glycan G0-N, in which a terminal N-acetylglucosamine (GlcNAc) is attached to either the alpha-3 or alpha-6 branch of the common N-glycan pentasaccharide core. We then use IR fingerprints of these newly identified isomers to identify the positional isomers of G1 and G1F, which are biantennary complex-type N-glycans with a terminal galactose attached to either the alpha-3 or alpha-6 branch, and in the case of G1F a fucose attached to the reducing-end GlcNAc. Starting with just a few analytical standards, this fragment-based spectroscopy method allows us to develop a database which we can use to identify positional isomers. The generalization of this approach would greatly facilitate glycan analysis.

Automated summary

Glycans, present on the surface of cells in all living organisms, play crucial roles in biological processes. However, the complexity of their isomeric structures poses challenges for structural characterization. Researchers have developed a method combining ion-mobility spectrometry, collision-induced dissociation, and cryogenic infrared spectroscopy to determine the structure of positional isomers of N-glycans. This approach enables the identification of previously difficult-to-obtain analytical standards and facilitates glycan analysis.