Background: Radical Probe Mass Spectrometry (RP-MS) describes a pioneering
methodology in structural biology that enables the study of protein structures, their interactions, and
dynamics on fast timescales (down to sub-milliseconds). Hydroxyl radicals (•OH) generated
directly from water within aqueous solutions induce the oxidation of reactive, solvent accessible
amino acid side chains that are then analyzed by mass spectrometry. Introduced in 1998 at the
American Society for Mass Spectrometry annual conference, RP-MS was first published on in
Objective: This review article describes developments and applications of the RP-MS methodology
over the past two decades.
Methods: The RP-MS method has been variously referred to as synchrotron X-ray radiolysis
footprinting, Hydroxyl Radical Protein Footprinting (HRPF), X-ray Footprinting with Mass
Spectrometry (XF-MS), Fast Photochemical Oxidation of Proteins (FPOP), oxidative labelling,
covalent oxidative labelling, and even the Stability of Proteins from Rates of Oxidation (SPROX).
Results: The article describes the utility of hydroxyl radicals as a protein structural probe, the
advantages of RP-MS in comparison to other MS-based approaches, its proof of concept using ion
mobility mass spectrometry, its application to protein structure, folding, complex and aggregation
studies, its extension to study the onset of protein damage, its implementation using a high
throughput sample loading approach, and the development of protein docking algorithms to aid
with data analysis and visualization.
Conclusion: RP-MS represents a powerful new structural approach that can aid in our
understanding of the structure and functions of proteins, and the impact of sustained oxidation on
proteins in disease pathogenesis.