Optimizing Hydroxyl Radical Footprinting Analysis of Biotherapeutics Using Internal Standard Dosimetry

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.