Radical Probe Mass Spectrometry (RP-MS)
In collaboration, our laboratory was the first to use radicals for beneficial effect to study the structures of proteins. Originating from radiolytic synchrotron protein footprinting studies using hydroxyl radicals, Radical Probe Mass Spectrometry involves the production of a high flux of hydroxyl and other oxygen radicals to react with proteins on short millisecond timescales to effect their limited oxidation. The original studies (below) found a strong correlation between the level of oxidation and the accessibility of amino acid side chains to the bulk solvent.
A measure of the oxidation rates or levels enables the surface of a protein to be monitored (surface mapping) including changes to that surface during folding and unfolding events and following the interaction of proteins with other macromolecules.
First reported in 1999, the approach has been applied by a growing number of research groups worldwide in applications in structural biology. See Chapter 6 of the book "Mass Spectrometry of Protein Interactions" for a full description of these events and the development of RP-MS.
Our laboratory was the first to apply it to study protein complexes (2003) and to extend it to study earlier onset protein oxidative damage by extending the reaction timescale (>50msec) (2005). We also developed the first protein docking algorithm specifically designed for RP-MS data (2006)
Original reference:
Maleknia SD, Chance MR, Downard KM (1999) Electrospray-Assisted Modification of Proteins. A Radical Probe of Protein Structure (lead article), Rapid Commun. Mass Spectrom. 13, 2352-2358.
First review of approach:
Maleknia S.D., Downard K.M. (2001) Radical Approaches to Probe Protein Structure, Folding and Interactions by Mass Spectrometry, Mass Spectrom. Rev., 20(6): 388-401.
First application to a protein complex:
Wong JWH, Maleknia SD, Downard K.M. (2003) Study of the RNase S-Protein S-Peptide Complex using a Radical Probe and Electrospray Ionization Mass Spectrometry, Anal. Chem. (accelerated article), 75: 1557-1563.
First extension of RP-MS to study early onset protein oxidative damage:
Shum W-K, Maleknia SD, Downard KM, (2005) Onset of Oxidative Damage in α-Crystallin by
Radical Probe Mass Spectrometry, Anal. Biochem., 344: 247-256.
First protein docking algorithm for use with RP-MS data:
Gerega SK, Downard KM (2006) PROXIMO - A Docking New Algorithm to Model Protein
Complexes Using Data from Radical Probe Mass Spectrometry, Bioinformatics, 22: 1702-1709.
Other references:
Maleknia SD, Ralston C, Kiselar JG, Downard KM, Chance MR (2001) Determination of
Macromolecular Folding and Structure through Synchrotron X-ray Footprinting Techniques, Anal.
Biochem., 289: 103-115.
Maleknia SD, Downard KM (2001) Unfolding of apomyoglobin helices by synchrotron radiolysis and mass spectrometry, Eur J Biochem. 268, 5578-5588.
Wong J.W.H., Maleknia S.D., Downard K.M. (2004) Photochemical and Electrophysical
Production of Radicals on Millisecond Timescales. A Probe of Protein Structure, Dynamics and Interactions, Photochem. Photobiol Sciences, 3, 741-748.
Wong JWH, Maleknia SD, Downard KM (2005) Hydroxyl Radical Probe of the Calmodulin-Melittin Complex Interface by Electrospray Ionization Mass Spectrometry, J. Am. Soc. Mass Spectrom., 16: 225-233.
Issa S, Downard KM (2006) Interaction between Alpha and Upsilon-Crystallin Common to the Eye of the Australian Platypus by Radical Probe Mass Spectrometry, J. Mass Spectrom., 41: 1298-1303.
Diemer H, Atmanene C, Sanglier S, Morrissey B, Van Dorsselaer A, Downard KM (2009)
Structural Features of the βB2-B3-crystallin heterodimer by Radical Probe Mass Spectrometry (RP-MS), J. Mass Spectrom., 44, 803-812.
