Sharp J.S; (2019) Am Pharmaceut Rev 22, 50-55
ABSTRACT
Protein higher order structure (HOS) analysis is one of the most complicated steps in biopharmaceutical analysis. Hydroxyl radical protein footprinting (HRPF) is a technology developed over the past three decades for characterizing protein higher order structure using mass spectrometry.1,2 Proteins are exposed to freely diffusing hydroxyl radicals in solutions, and these hydroxyl radicals modify amino acid side chains based, in part, on their solvent accessibility. The amount of side chain oxidation is measured by proteolysis followed by liquid chromatography coupled to mass spectrometry (LC-MS). By comparing the footprint of a protein sequence in two different structural states (e.g. two different formulations; ligandbound vs. ligand-free; glycosylated vs. deglycosylated; etc.), changes in the protein topography can be detected and localized based on changes in the amount of oxidation of a given section of a protein. This information can be used to determine antibody epitopes or ligand binding sites, regions of instability or aggregation interfaces, proteinprotein interaction surfaces, sites of allostery, or answer numerous other HOS questions with no theoretical limitations on analyte size and tremendous flexibility on sample matrix. As one example, HRPF by FPOP was used to identify the broadly neutralizing b12 antibody epitope in fully glycosylated gp120 of HIV (Figure 1).3 This represented Figure 1. a very challenging problem in HOS characterization, due to the flexibility of the gp120 variable regions and the high N-linked glycan content (~50% of the total glycoprotein mass). While many benchtop methods for HRPF have been reported, they all share similar concerns that must be addressed in experimental design: maintaining native structure throughout the labeling process; preventing secondary oxidation; measuring and compensating for differential radical production and scavenging in your sample; and understanding the limitations of data analysis and interpretation. This article discusses these critical concepts along with their impact on HRPF results, focusing on a popular benchtop method for HRPF radical generation: Fast Photochemical Oxidation of Proteins (FPOP).4 When these factors are understood and controlled, HRPF generates unique and sensitive HOS information in a highly flexible platform, and adds considerable value when integrated into existing HOS analysis workflows.