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

J Am Soc Mass Spectrom. 2005 Dec;16(12):2057-63. doi: 10.1016/j.jasms.2005.09.008. Epub 2005 Nov 2.


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 approximately 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.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / analysis
  • Amino Acids / chemistry*
  • Amino Acids / radiation effects
  • Apoproteins / analysis*
  • Apoproteins / chemistry*
  • Gas Chromatography-Mass Spectrometry / methods*
  • Hydrogen Peroxide / chemistry*
  • Hydrogen Peroxide / radiation effects
  • Lasers*
  • Myoglobin / analysis*
  • Myoglobin / chemistry*
  • Oxidation-Reduction
  • Photolysis / radiation effects
  • Solvents
  • Time Factors


  • Amino Acids
  • Apoproteins
  • Myoglobin
  • Solvents
  • apomyoglobin
  • Hydrogen Peroxide