Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry.
Protein surfaces are important in most biological processes, including protein-protein interactions, enzymatic catalysis, and protein-ligand binding. We report a method in which hydroxyl radicals generated by a rapid-UV irradiation of a 15% hydrogen peroxide solution were utilized to oxidize specific amino acid side chains of two model proteins (lysozyme, beta-lactoglobulin A), according to the residues' chemical reactivities and the solvent accessibility of the reactive carbons and sulfurs in the residue. Oxidized peptides generated by tryptic digestion were identified by electrospray-Fourier transform mass spectrometry. The specific sites of the stable modification were then identified by reverse-phase liquid chromatography coupled to quadropole ion trap tandem mass spectrometry. The solvent accessibility of the residue was shown to directly affect the rate of oxidation by this method (with the exception of methionine), supporting its use as a rapid measure of the solvent accessibility of specific residues, and in some cases, individual atoms.
Sharp
Joshua S
JS
Graduate School of Genome Science and Technology, The University of Tennessee-Oak Ridge National Laboratory, 1060 Commerce Park, Oak Ridge, TN 37830-8026, USA.
Becker
Jeffrey M
JM
Hettich
Robert L
RL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002645:Chickens; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D005583:Fourier Analysis; D006861:Hydrogen Peroxide / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry; D007782:Lactoglobulins / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010777:Photochemistry; D012997:Solvents / Q000737:chemistry*; D013499:Surface Properties; D014357:Trypsin; D014466:Ultraviolet Rays
D000596:Amino Acids; D007782:Lactoglobulins; D012997:Solvents; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; C421864:hen egg lysozyme; D009113:Muramidase; D014357:Trypsin