Hydroxyl radical footprinting-mass spectrometry (HRF-MS) is a powerful technique for measuring protein structure by quantitating the solvent accessibility of amino acid side-chains; and when used in comparative analysis, HRF-MS data can provide detailed information on changes in protein structure. However, consistently controlling the amount of hydroxyl radical labeling of a protein requires the precise understanding of both the amount of radicals generated and half-life of the radicals in solution. The latter is particularly important for applications such as protein-protein and protein-ligand interactions, which may have different characteristics such as intrinsic reactivity and buffer components, and can cause differences in radical scavenging (herein termed "scavenging potential") between samples. To address this inherent challenge with HRF-MS analysis, we describe the comprehensive implementation of an internal standard (IS) dosimeter peptide leucine enkephalin (LeuEnk) for measuring the scavenging potential of pharmaceutically relevant proteins and formulation components. This further enabled evaluation of the critical method parameters affecting the scavenging potential of samples subjected to HRF-MS using fast photochemical oxidation of proteins. We demonstrate a direct correlation between the oxidation of the IS peptide and biotherapeutic target proteins, and show the oxidation of the IS can be used as a guide for ensuring equivalent scavenging potentials when comparing multiple samples. Establishing this strategy enables optimization of sample parameters, a system suitability approach, normalization of data, and comparison/harmonization of HRF-MS analysis across different laboratories.

中文翻译：

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使用内标剂量法优化生物治疗药物的羟基自由基足迹分析。

羟自由基足迹质谱法（HRF-MS）是通过定量氨基酸侧链的溶剂可及性来测量蛋白质结构的强大技术。当用于比较分析时，HRF-MS数据可以提供有关蛋白质结构变化的详细信息。但是，要始终如一地控制蛋白质中羟基自由基标记的数量，就需要对自由基产生的数量和溶液中自由基的半衰期都有准确的了解。后者对于诸如蛋白质-蛋白质和蛋白质-配体相互作用之类的应用特别重要，这些应用可能具有不同的特征，例如固有的反应性和缓冲液成分，并可能导致样品之间的自由基清除（本文称为“清除潜力”）方面的差异。为了通过HRF-MS分析解决这一固有挑战，我们描述了一种内标（IS）剂量计肽亮氨酸脑啡肽（LeuEnk）的全面实施方法，用于测量药物相关蛋白质和制剂组分的清除潜能。这使得能够使用蛋白质的快速光化学氧化方法评估影响HRF-MS样品清除潜力的关键方法参数。我们证明了IS肽的氧化与生物治疗靶蛋白之间的直接相关性，并显示了IS的氧化可作为比较多个样品时确保等效清除潜能的指南。建立此策略可以优化样本参数，系统适用性方法，数据规范化，