Extensive Investigations on Oxidized Amino Acid Residues in H2O2-Treated Cu,Zn-SOD Protein with LC-ESI-Q-TOF-MS, MS/MS for the Determination of the Copper-Binding Site
- 23 August 2001
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 123 (38) , 9268-9278
- https://doi.org/10.1021/ja015953r
Abstract
The ESI (electrospray ionization)-Q-TOF (tandem quadrupole/orthogonal-acceleration time-of-flight) mass spectrometer combined with the nano-HPLC (high performance liquid chromatography) system was utilized to pinpoint the Cu-binding site in Cu,Zn-SOD (superoxide dismutase) protein. Cu,Zn-SOD was treated with hydrogen peroxide, intended to specifically oxidize histidine residues coordinated to the copper ion as a mass spectrometric probe. The oxidized Cu,Zn-SOD was then fragmented with the successive treatment of endoproteinase Asp-N and DTT (dithiothreitol). Separation of the peptide mixture with the nano-HPLC and the on-line ESI-Q-TOF MS analysis revealed that only two peptide fragments were oxidized to a significant extent. Further analyses of oxidized peptide fragments with LC-ESI-Q-TOF-MS/MS disclosed that three out of four Cu-coordinated histidine residues were specifically oxidized by action of a redox-active copper ion and hydrogen peroxide, demonstrating the copper-catalyzed oxidation of amino acid ligands could be a versatile tool for the mass spectrometric determination of the copper-binding site. In addition, proline and valine residues in the proximity of the Cu ion were found to be oxidized upon H2O2 treatment.Keywords
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