Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry.
A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the surface of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions with synchrotron light for several milliseconds result in the reaction of amino acid side chains. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side chains to the bulk solvent. The aromatic and sulfur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for each of the proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates have been found to correlate closely with a theoretical measure of the accessibility of residue side chains to the bulk solvent in the native protein structure. The synchrotron-based approach is able to distinguish the relative accessibility of the tryptophan residue side chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues based on their rates of oxidation.
Maleknia
Simin D
SD
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461-1602, USA.
Kiselar
Janna G
JG
Downard
Kevin M
KM
D000595:Amino Acid Sequence; D017665:Hydroxyl Radical / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry* / Q000528:radiation effects; D010084:Oxidation-Reduction; D011854:Radiochemistry; D015203:Reproducibility of Results; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013499:Surface Properties; D017356:Synchrotrons
D012997:Solvents; D017665:Hydroxyl Radical; D008715:Methionine; D009113:Muramidase