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

The ESI (electrospray ionization)-Q-TOF (tandem quadrupole/orthogonal-accel eration 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 tre ated with hydrogen peroxide, intended to specifically oxidize histidine res idues coordinated to the copper ion as a mass spectrometric probe. The oxid ized Cu,Zn-SOD was then fragmented with the successive treatment of endopro teinase Asp-N and DTT (dithiothreitol). Separation of the peptide mixture w ith 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 analys es of oxidized peptide fragments with LC-ESI-Q-TOF-MS/MS disclosed that thr ee out of four Cu-coordinated histidine residues were specifically oxidized by action of a redox-active copper ion and hydrogen peroxide, demonstratin g 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 wer e found to be oxidized upon H2O2 treatment.