Abstract
We report here the novel use of electrochemistry to generate covalent oxidative labels on intact proteins in both non-native and physiologically relevant solutions as a surface mapping probe of higher order protein structure. Two different electrodes were tested across a range of experimental parameters including voltage, flow rate, and solution electrolyte composition to affect the extent of oxidation on intact proteins, as measured both on-line and off-line with mass spectrometry. Oxidized proteins were collected off-line for proteolytic digestion followed by LC-MS/MS analysis. Peptide MS/MS data were searched with the InsPecT scoring algorithm for forty-six oxidative mass shifts previously reported in the literature. Preliminary data showed agreement between solvent accessibility and the oxidation status of many ubiquitin residues in aqueous buffer, while more buried residues were found to be oxidized in non-native solution. Our results indicate that electrochemical oxidation using a boron-doped diamond electrode has the potential to become a useful and easily accessible tool for conducting oxidative surface mapping experiments.
