title: Advances in radical probe mass spectrometry for protein footprinting in chemical biology applications.
abstract: Radical Probe Mass Spectrometry (RP-MS), first introduced in 1999, utilizes hydroxyl radicals generated directly within aqueous solutions using synchrotron radiolysis, electrical discharge, and photochemical laser sources to probe protein structures and their interactions. It achieves this on millisecond and submillisecond timescales that can be used to capture protein dynamics and folding events. Hydroxyl radicals are ideal probes of solvent accessibility as their size approximates a water molecule. Their high reactivity results in oxidation at a multitude of amino acid side chains providing greater structural information than a chemical cross-linker that reacts with only one or few residues. The oxidation of amino acid side chains occurs at rates in accord with the solvent accessibility of the residue so that the extent of oxidation can be quantified to reveal a three-dimensional map or footprint of the protein's surface. Mass spectrometry is central to this analysis of chemical oxidative labelling. This tutorial review, some 15 years on from the first reports, highlights the development and significant growth of the application of RP-MS including its validation and utility with ion-mobility mass spectrometry (IM-MS), the use of RP-MS data to help model protein complexes, studies of the onset of oxidative damage, and more recent advances that enable high throughput applications through simultaneous protein oxidation and on-plate deposition. The accessibility of the RP-MS technology, by means of a modified electrospray ionization source, enables the approach to be implemented in many laboratories to address a wide range of applications in chemical biology.
authors.affiliation: School of Civil and Environmental Engineering, University of New South Wales, Sydney, Australia.
authors.forename: Simin D
authors.initials: SD
authors.lastname: Maleknia
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Downard
mesh_terms: D000596:Amino Acids; D001671:Biochemistry / Q000379:methods*; D006878:Hydroxides; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D011487:Protein Conformation; D017510:Protein Folding; D023302:Protein Footprinting / Q000379:methods*
chemical_list: D000596:Amino Acids; D006878:Hydroxides; C031356:hydroxide ion