The use of hydroxyl radicals to covalently label the solvent-exposed surface of proteins has been shown to be a powerful tool to examine the structure of proteins and intermolecular interfaces. Current methods to generate hydroxyl radicals for footprinting experiments rely on the laser photolysis of H2O2 or the synchrotron radiolysis of water, which adds significant costs and/or complexity to the experiments. In this work, we develop the electro-Fenton reaction as a means to generate hydroxyl radicals for structural footprinting mass spectrometry experiments to complement current laser and synchrotron-based methods, while reducing the costs and complexity of initiating such experiments. The use of an electrochemical flow cell also enables control of the timing and extent of the radical generation process, while reducing the complexity typically associated with radical footprinting experiments. Ubiquitin, a model protein, was labeled with electro-Fenton generated hydroxyl radicals and top-down proteomics was used to verify oxidation sites on the protein surface.
ASJC Scopus subject areas
- Analytical Chemistry