Extensive investigations on oxidized amino acid residues in H(2)O(2)-treated Cu,Zn-SOd protein with LC-ESI-Q-TOF-MS, MS/MS for the determination of the copper-binding site.
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 H(2)O(2) treatment.
Kurahashi
T
T
Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Miyazaki
A
A
Suwan
S
S
Isobe
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D003300:Copper; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase