Yusuph Quadrat, Misra Sandeep K, Liu Hao, Sharp Joshua S
Department of Chemistry and Biochemistry, University of Mississippi, University, Mississippi 38677, United States.
Department of Biomolecular Sciences, University of Mississippi, University, Mississippi 38677, United States.
bioRxiv. 2025 May 10:2025.05.06.652515. doi: 10.1101/2025.05.06.652515.
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) shows no significant change in modification in non-binding protein solutions. However, in the presence of two proteins that bind NAG specifically, NAG 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.
碳水化合物以多种形式存在于生物体内，既包括独立的聚糖，也包括糖缀合物，如糖蛋白、糖脂和糖胺聚糖。这些结构在许多生物过程中发挥着关键作用，通常由碳水化合物与其他生物分子的相互作用介导或影响。然而，由于碳水化合物的结构复杂性、其动态构象行为以及通常涉及的低结合亲和力，研究这些相互作用具有特别的挑战性。为了应对这些挑战，我们正在开发一种新方法，该方法利用基于质谱的碳水化合物自由基足迹法（RFC）。我们通过测量羟基自由基和三氟甲基自由基介导的氧化表观速率的变化，监测寡糖内特定区域的溶剂可及性变化。在我们的研究中，一组三糖异构体和N，N'，N''-三乙酰壳三糖（NAG）在非结合蛋白溶液中的修饰没有显著变化。然而，在存在两种特异性结合NAG的蛋白质时，NAG氧化减少。我们发现，在共价标记条件下，游离还原端是羟基自由基氧化的主要位点，这使其能够区分聚糖还原端处的相互作用。相反，三氟甲基自由基通过取代C-H键在整个三糖上广泛标记。总体而言，这种方法为识别聚糖-蛋白质相互作用和绘制聚糖的结合界面提供了一种强大的新方法。
