Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes.
Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes.
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DOI：
10.3791/61580
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发表时间：
2020-09-01
期刊：
影响因子：
--
通讯作者：
Sharp JS
中科院分区：
文献类型：
--
作者：
Misra SK;Sharp JS
蛋白质快速光化学氧化 (FPOP) 是一种基于质谱的结构生物学技术，可探测蛋白质的溶剂可及表面积。该技术依赖于氨基酸侧链与在溶液中自由扩散的羟基自由基的反应。 FPOP 通过激光光解过氧化氢原位产生这些自由基，产生大量羟基自由基，并在一微秒的时间内耗尽。当这些羟基自由基与溶剂可及的氨基酸侧链反应时，反应产物表现出质量转移，可以通过质谱法测量和定量。由于氨基酸的反应速率部分取决于该氨基酸的平均溶剂可及表面，因此测量到的蛋白质给定区域氧化量的变化可以与该区域在不同构象之间的溶剂可及性变化直接相关（例如配体结合与无配体、单体与聚集体等）。FPOP 已应用于生物学中的许多问题，包括蛋白质-蛋白质相互作用、蛋白质构象变化和蛋白质-配体结合。由于羟基自由基的可用浓度根据 FPOP 实验中的许多实验条件而变化，因此监测蛋白质分析物所暴露的有效自由基剂量非常重要。通过结合在线剂量计来测量 FPOP 反应的信号，并实时调整激光能量密度以实现所需的氧化量，可以有效地实现这种监测。通过这种补偿，可以使用相对较低的样品量来确定异质样品中反映构象变化、配体结合表面和/或蛋白质-蛋白质相互作用界面的蛋白质形貌的变化。蛋白质的快速光化学氧化是蛋白质结构表征的新兴技术。不同的溶剂添加剂和配体具有不同的羟基自由基清除性能。为了比较不同条件下的蛋白质结构，需要实时补偿反应中产生的羟基自由基以标准化反应条件。
Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g. ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts. Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.
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