Abstract
Carbohydrates are found in various forms in living organisms, both as free-standing glycans as well as glycoconjugates including glycoproteins, glycolipids, and glycosaminoglycans. These structures play crucial roles in many biological processes, often mediated or influenced by interactions of carbohydrates with other biomolecules. However, studying these interactions is particularly challenging due to the structural complexity of carbohydrates, their dynamic conformational behavior, and the low binding affinities often involved. To address these challenges, we are developing a novel method that leverages mass spectrometry-based radical footprinting of carbohydrates (RFC). We monitored changes in the solvent accessibility of specific regions within oligosaccharides by measuring variations in the apparent rate of hydroxyl radical and trifluoromethyl radical-mediated oxidation. In our studies, a collection of trisaccharide isomers and N, N',N''-triacetylchitotriose (NAG 3 ) shows no significant change in modification in non-binding protein solutions. However, in the presence of two proteins that bind NAG 3 specifically, NAG 3 oxidation is reduced. We find that the free reducing end is the primary site of hydroxyl radical oxidation under covalent labeling conditions, allowing it to distinguish interactions at the glycan reducing end. Trifluoromethyl radicals, conversely, label broadly across the trisaccharide by substitution into a C-H bond. Overall, this approach offers a powerful new approach for identifying glycan-protein interactions and mapping the binding interface of glycans.