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Rp-MS / PROTEIN FOOTPRINTING

Radical Probe Mass Spectrometry (RP-MS)   (also Hydroxyl Radical Protein Footprinting)

 

In collaboration, our laboratory was the first to use radicals for beneficial effect to study the structures of proteins and their complexes.

 

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 in what has also become known as structural proteomics.

 

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), employed ion mobility with RP-MS for the first time (2011), and provided the first direct evidence of the preservation of structural integrity upon limited oxidation using ion mobility mass spectrometry (2012). See the recent reviews below for a true history of these events.

 

Recent reviews:

 

Downard KM, Maleknia SD (2018) Mass Spectrometry in Structural Proteomics: The Case for Radical Probe Protein Footprinting, Trends in Anal, Chem., 110: 293-302.

 

Maleknia SD, Downard KM (2019) Protein Footprinting with Radical Probe Mass Spectrometry - Two Decades of Achievement, in Current Perspectives on Footprinting as a Tool for Structural Biology (Special Issue ed. Ralston CY), Prot. Pep. Lett., 26(1): 4-15.

 

Maleknia S.D., Downard K.M. (2014) Advances in Radical Probe Mass Spectrometry for Protein Footprinting in Chemical Biology Applications, Chem. Soc. Rev., 43: 3244-3258.

 

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.

 

Other RP-MS / Protein Footprinting firsts 

 

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 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 / protein footprinting data:

 

Gerega SK, Downard KM (2006) PROXIMO - A New Docking Algorithm to Model Protein Complexes Using Data from Radical Probe Mass Spectrometry, Bioinformatics, 22: 1702-1709.

 

First application to study protein complexes in concert with Ion Mobility MS:

 

Downard KM, Kokabu Y, Ikeguchi M, Akashi S (2011) Homology Modelled Structure of the Beta-B2B3-Crystallin Heterodimer Studied by Ion Mobility and Radical Probe Mass Spectrometry, FEBSJ, 278, 4044-4054.

 

Akashi S, Maleknia SD, Saikusa K, Downard KM (2015) Stability of the βB2B3 Crystallin Heterodimer to Increased Oxidation by Radical Probe and Ion Mobility Mass Spectrometry, J. Struct. Biol., 189: 1-8.

 

First direct evidence of structural integrity upon limited oxidation by Ion Mobility MS:

 

Downard KM, Maleknia SD, Akashi S (2012) Impact of Limited Oxidation on Protein Ion Mobility and Structure of Importance to Footprinting by Radical Probe Mass Spectrometry, Rapid Commun. Mass Spectrom., 26, 226-230.

 

First coupling of approach to MALDI deposition for high-throughput analysis:

 

Maleknia SD, Downard KM (2012) On-plate deposition of oxidized proteins to facilitate protein footprinting studies by radical probe mass spectrometry, Rapid Commun Mass Spectrom. 26: 2311-2318.

 

Early reviews:

 

Maleknia SD, Downard KM (2007) Genesis and Application of Radical Probe Mass Spectrometry (RP‐MS) to Study Protein Interactions, in Mass Spectrometry of Protein Interactions, pp.109 - 133, Wiley, New Jersey USA.      DOI: 10.1002/9780470146330.ch6

 

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.

 

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.

 

Other references on protein folding and interactions:

 

Maleknia SD, Downard KM (2001) Unfolding of apomyoglobin helices by synchrotron radiolysis and mass spectrometry, Eur J Biochem. 268, 5578-5588. 

 

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.