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 specifi...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2004-02, Vol.76 (3), p.672-683
Main Authors: Sharp, Joshua S, Becker, Jeffrey M, Hettich, Robert L
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acids - chemistry
Analytical biochemistry: general aspects, technics, instrumentation
Analytical chemistry
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Cattle
Chemistry
Chickens
Chromatographic methods and physical methods associated with chromatography
Chromatography
Chromatography, Liquid
Crystallography, X-Ray
Exact sciences and technology
Fourier Analysis
Fundamental and applied biological sciences. Psychology
Hydrogen Peroxide - chemistry
Hydroxyl Radical - chemistry
Lactoglobulins - chemistry
Mass Spectrometry - methods
Models, Molecular
Molecular Sequence Data
Muramidase - chemistry
Other chromatographic methods
Oxidation
Oxidation-Reduction
Peptides
Photochemistry
Proteins
Solvents
Solvents - chemistry
Spectrometric and optical methods
Surface Properties
Trypsin
Ultraviolet Rays
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Summary: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, β-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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac0302004