LAUSR

Glycans attached to lipids and membrane-bound and secreted proteins and peptides mediate many important physiological and pathophysiological processes through interactions with glycan-binding proteins (GBPs). However, uncovering functional glycan ligands is challenging due to the large number of naturally occurring glycan structures, the limited availability of glycans in their purified form, the low affinities of GBP-glycan interactions, and limitations in existing binding assays. This work explores the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening libraries of N-glycans derived from natural sources. The assay was tested by screening a small defined library of complex N-glycans, at equimolar concentrations, against plant and human GBPs with known specificities for either α2-3- or α2-6-linked sialosides, with affinities in the mM to μM range. Validation experiments, performed in negative ion mode, revealed that bound N-glycan ligands are readily released, as intact deprotonated ions, from GBPs in the gas phase using collision-induced dissociation. Moreover, the relative abundances of the released ligands closely match their solution affinities. Results obtained for a natural N-glycan library produced from cultured immune cells serve to highlight the ease with which CaR-ESI-MS can screen complex mixtures of N-glycan for interactions. Moreover, scaling the relative abundances of released glycan ligands according to their relative abundances in solution, as determined by hydrophilic interaction-ultra high-performance liquid chromatography of the fluorescently-labeled library, allows the relative affinities of glycan ligands to be ranked.