CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2022 Jan 18;94(2):1135-1142. doi: 10.1021/acs.analchem.1c04209. Epub 2021 Dec 29.
Ultraviolet (UV) laser photolysis of hydrogen peroxide (HO) for the 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.
通过过氧化氢（HO）的紫外线（UV）激光光解来生成羟基自由基（OH），是一种广泛应用于天然蛋白质的氧化足迹分析和基于质谱（MS）的结构分析的策略。然而，实现无过氧化物的光化学氧化足迹分析仍然具有挑战性。在此，我们描述了通过氯介导的无过氧化物的蛋白质光化学氧化（PPOP）来对天然蛋白质进行足迹分析。在含有 10 mM Gln 的生物相容性磷酸盐缓冲盐水（PBS）中制备蛋白质样品，作为自由基清除剂，并在单脉冲（10 ns）193 nm ArF UV 激光照射下直接在毛细管流反应器中进行氧化。主要氧化的蛋白质残基为 CMYWFHLI。我们证明，PPOP-MS 策略对蛋白质的高级结构非常敏感，可用于监测蛋白质-药物界面，为蛋白质结构-功能研究提供了一种很有前途的足迹分析替代方法。
