Protein Analytical Chemistry, Genentech Inc., South San Francisco, 1 DNA Way, South San Francisco, California 94080, United States.
Late Stage Pharmaceutical Development, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States.
J Am Soc Mass Spectrom. 2020 Jul 1;31(7):1563-1571. doi: 10.1021/jasms.0c00146. Epub 2020 May 27.
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.
羟基自由基足迹质谱（HRF-MS）是一种通过定量测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。然而，要始终如一地控制蛋白质的羟基自由基标记量，就需要精确地了解自由基的生成量和自由基在溶液中的半衰期。后者对于蛋白质-蛋白质和蛋白质-配体相互作用等应用特别重要，因为这些应用可能具有不同的特性，如固有反应性和缓冲成分，并且可能导致样品之间的自由基清除（在此称为“清除潜力”）存在差异。为了解决 HRF-MS 分析中固有的这一挑战，我们全面实施了内标（IS）示踪剂亮氨酸脑啡肽（LeuEnk），用于测量药物相关蛋白质和制剂成分的清除潜力。这进一步使我们能够评估使用蛋白质快速光化学氧化法影响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存在直接相关性，并表明 IS 的氧化可作为指导，确保在比较多个样品时具有等效的清除潜力。建立这种策略可以优化样品参数、系统适用性方法、数据归一化以及跨不同实验室的 HRF-MS 分析比较/协调。
