Using microwave-assisted metal-catalyzed oxidation reactions and mass spectrometry to increase the rate at which the copper-binding sites of a protein are determined

Controlled microwave irradiation has been used to accelerate metal-catalyzed oxidation (MCO) reactions that site-specifically oxidize the amino acids bound to Cu in a metalloprotein. When combined with mass spectrometry, these MCO reactions provide a sensitive method for determining Cu-protein binding sites. In this work, we demonstrate that microwave irradiation can increase the rate of these site-specific oxidation reactions for Cu/Zn superoxide dismutase by at least 15-fold. By choosing the appropriate microwave power, changes to the protein's structure can be avoided while still accelerating the oxidation reactions. The maximum microwave power suitable for maintaining the protein's structural integrity can be readily determined by measuring the oxidation extent of different peptide fragments as a function of microwave power. Such determinations require several measurements and thus limit the overall throughput of this approach; however, these microwave-assisted reactions do provide good time resolution for studying dynamic changes to Cu-binding sites.