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Protein Footprinting with Radical Probe Mass Spectrometry- Two Decades of Achievement

[ Vol. 26 , Issue. 1 ]


Simin D. Maleknia* and Kevin M. Downard   Pages 4 - 15 ( 12 )


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 1999.

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.


Protein footprinting, hydroxyl radical, radical probe, mass spectrometry, protein structure, protein interaction, oxidative damage, protein aggregates.


School of Mathematical and Physical Sciences, University of Technology Sydney, Sydney, NSW, Infectious Disease Responses Laboratory, University of New South Wales-Medicine, Sydney, NSW

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