Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale


Footprinting of proteins by hydroxyl radicals generated on the millisecond to minute timescales to probe protein surfaces suffers from the uncertainty that radical reactions cause the protein to unfold, exposing residues that are protected in the native protein. To circumvent this possibility, we developed a method using a 248 nm KrF excimer laser to cleave hydrogen peroxide at low concentrations (15 mM, 0. 04%), affording hydroxyl radicals that modify the protein in less than a microsecond. In the presence of a scavenger (20 mM glutamine), the radical lifetimes decrease to ∼1 microsecond, yet the reaction timescales are sufficient to provide significant oxidation of the protein. These times are arguably faster than super-secondary protein structure can unfold as a result of the modification. The radical formation step takes place in a nanoliter flow cell so that only one laser pulse irradiates each bolus of sample. The oxidation sites are located using standard analytical proteomics, requiring less than a nanomole of protein. We tested the method with apomyoglobin and observed modifications in accord with solvent accessibility data obtained from the crystal structure of holomyoglobin. Additionally, the results indicate that the F-helix is conformationally flexible in apomyoglobin, in accord with NMR results. We also find that the binding pocket is resistant to modifications, indicating that the protein pocket closes in the absence of the heme group—conclusions that cannot be drawn from current structural methods. When developed further, this method may enable the determination of protein-ligand interfaces, affinity constants, folding pathways, and regions of conformational flexibility.


  1. 1.

    Zhu, M. M.; Rempel, D. L.; Du, Z.; Gross, M. L. Quantification of Protein-Ligand Interactions by Mass Spectrometry, Titration, and H/D Exchange: PLIMSTEX. J. Am. Chem. Soc. 2003, 125, 5252–5253.

    Article  CAS  Google Scholar 

  2. 2.

    Maleknia, S. D.; Ralston, C. Y.; Brenowitz, M. D.; Downard, K. M.; Chance, M. R. Determination of Macromolecular Folding and Structure by Synchrotron X-Ray Radiolysis Techniques. Anal. Biochem. 2001, 289, 103–115.

    Article  CAS  Google Scholar 

  3. 3.

    Kiselar, J. G.; Maleknia, S. D.; Sullivan, M.; Downard, K. M.; Chance, M. R. Hydroxyl Radical Probe of Protein Surfaces Using Synchrotron X-Ray Radiolysis and Mass Spectrometry. Int. J. Radiat. Biol. 2002, 78, 101–114.

    Article  CAS  Google Scholar 

  4. 4.

    Sharp, J. S.; Becker, J. M.; Hettich, R. L. Analysis of Protein Solvent Accessible Surfaces by Photochemical Oxidation and Mass Spectrometry. Anal. Chem. 2004, 76, 672–683.

    Article  CAS  Google Scholar 

  5. 5.

    Maleknia, S. D.; Chance, M. R.; Downard, K. M. Electrospray-Assisted Modification of Proteins: A Radical Probe of Protein Structure. Rapid Commun. Mass Spectrom. 1999, 13, 2352–2358.

    Article  CAS  Google Scholar 

  6. 6.

    Guan, J.-Q.; Takamoto, K.; Almo, S. C.; Reisler, E.; Chance, M. R. Structure and Dynamics of the Actin Filament. Biochemistry 2005, 44, 3166–3175.

    Article  CAS  Google Scholar 

  7. 7.

    Maleknia, S. D.; Wong, J. W. H.; Downard, K. M. Photochemical and Electrophysical Production of Radicals on Millisecond Timescales to Probe the Structure, Dynamics, and Interactions of Proteins. Photochem. Photobiol. Sci. 2004, 3, 741–748.

    Article  CAS  Google Scholar 

  8. 8.

    Naganathan, A. N.; Munoz, V. Scaling of Folding Times with Protein Size. J. Am. Chem. Soc. 2005, 127, 480–481.

    Article  CAS  Google Scholar 

  9. 9.

    Aye, T. T.; Low, T. Y.; Sze, S. K. Nanosecond Laser-Induced Photochemical Oxidation Method for Protein Surface Mapping with Mass Spectrometry. Anal. Chem. 2005, 77, 5814–5822.

    Article  CAS  Google Scholar 

  10. 10.

    Smith, D. L.; Dharmasiri, K. Protein-Ligand Binding Studied by Amide Hydrogen Exchange and Mass Spectrometry. NATO ASI Ser. C 1998, 510, 45–58.

    CAS  Google Scholar 

  11. 11.

    Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. Critical Review of Rate Constants for Reactions of Hydrated Electrons, Hydrogen Atoms, and Hydroxyl Radicals (.OH/.O−) in Aqueous Solution. J. Phys. Chem. Ref. Data 1988, 17, 513–886.

    Article  CAS  Google Scholar 

  12. 12.

    Masuda, T.; Nakano, S.; Kondo, M. Rate Constants for the Reactions of OH Radicals with the Enzyme Proteins as Determined by the p-Nitrosodimethylaniline Method. J. Radiat. Res. (Tokyo) 1973, 14, 339–345.

    Article  CAS  Google Scholar 

  13. 13.

    Xu, G.; Takamoto, K.; Chance, M. R. Radiolytic Modification of Basic Amino Acid Residues in Peptides: Probes for Examining Protein-Protein Interactions. Anal. Chem. 2003, 75, 6995–7007.

    Article  CAS  Google Scholar 

  14. 14.

    Gilmanshin, R.; Williams, S.; Callender, R. H.; Woodruff, W. H.; Dyer, R. B. Fast Events in Protein Folding: Relaxation Dynamics of Secondary and Tertiary Structure in Native Apomyoglobin. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 3709–3713.

    Article  CAS  Google Scholar 

  15. 15.

    Williams, S.; Causgrove, T. P.; Gilmanshin, R.; Fang, K. S.; Callender, R. H.; Woodruff, W. H.; Dyer, R. B. Fast Events in Protein Folding: Helix Melting and Formation in a Small Peptide. Biochemistry 1996, 35, 691–697.

    Article  CAS  Google Scholar 

  16. 16.

    Vu, D. M.; Myers, J. K.; Oas, T. G.; Dyer, R. B. Probing the Folding and Unfolding Dynamics of Secondary and Tertiary Structures in a Three-Helix Bundle Protein. Biochemistry 2004, 43, 3582–3589.

    Article  CAS  Google Scholar 

  17. 17.

    Eliezer, D.; Wright, P. E. Is Apomyoglobin a Molten Globule? Structural Characterization by NMR. J. Mol. Biol. 1996, 263, 531–853.

    Article  CAS  Google Scholar 

  18. 18.

    Scholes, G.; Shaw, P.; Willson, R. L.; Ebert, M. In Pulse Radiolysis, Ebert, M.; Keene, J. P.; Swallow, A. J.; Baxendale, J. H., Esd.; Academic Press: New York, 1965; pp. 151–164.

    Google Scholar 

  19. 19.

    Ge, Y.; Lawhorn, B. G.; ElNaggar, M.; Sze, S. K.; Begley, T. P.; McLafferty, F. W. Detection of Four Oxidation Sites in Viral Prolyl-4-Hydroxylase by Top-Down Mass Spectrometry. Protein Sci. 2003, 12, 2320–2326.

    Article  CAS  Google Scholar 

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Correspondence to Michael L. Gross.

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Published online November 2, 2005

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Hambly, D.M., Gross, M.L. Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale. J Am Soc Mass Spectrom 16, 2057–2063 (2005).

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  • Solvent Accessibility
  • Solvent Exposure
  • Laser Flash Photolysis
  • Oxidation Site
  • Unmodified Peptide