Chloride-Mediated Peroxide-Free Photochemical Oxidation of Proteins (PPOP) in Mass Spectrometry-Based Structural Analysis

Ultraviolet (UV) laser photolysis of hydrogen peroxide (H2O2) for the in situ generation of hydroxyl radicals (•OH) is a widely utilized strategy in the oxidation footprinting of native proteins and mass spectrometry (MS)-based structural analysis. However, it remains challenging to realize peroxide-free photochemical oxidation footprinting. Herein, we describe the footprinting of native proteins by chloride-mediated peroxide-free photochemical oxidation of proteins (PPOP). The protein samples are prepared within biocompatible phosphate-buffered saline (PBS) containing 10 mM Gln as radical scavengers and oxidized in a capillary flow reactor directly under a single-pulse (10 ns) irradiation of a 193 nm ArF UV laser. The main oxidized protein residues are CMYWFHLI. We demonstrate that the PPOP-MS strategy is highly sensitive to the protein high-order structures and can be applied to monitor the protein–drug interfaces, which provides a promising footprinting alternative for protein structure–function explorations.紫外线（UV）激光光解过氧化氢（H₂O₂）以原位生成羟基自由基（•OH），是天然蛋白质氧化足迹法与基于质谱（MS）的结构分析领域中广泛应用的策略。然而，实现无过氧化氢的光化学氧化足迹法仍颇具挑战。在此，我们报道了一种基于氯离子介导的无过氧化氢蛋白质光化学氧化（PPOP）的天然蛋白质足迹法。蛋白质样品于含10 mM谷氨酰胺（Gln，自由基清除剂）的生物相容性磷酸盐缓冲液（PBS）中制备，并在毛细管流动反应器中，直接经受193 nm ArF紫外激光的单脉冲（10 ns）辐照实现氧化。主要被氧化的蛋白质残基为CMYWFHLI。我们证实，PPOP-MS策略对蛋白质高级结构具有极高敏感性，可用于监测蛋白质-药物相互作用界面，为蛋白质结构-功能探索提供了一种极具潜力的足迹法替代方案。