Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

Abstract

Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent – hydroxyl radicals – for these measurements; however, many of these approaches require use of radioactive sources or caustic oxidizing chemicals. The purpose of this research was to evaluate and optimize the use of boron-doped diamond (BDD) electrochemistry as a highly accessible tool for producing hydroxyl radicals as a means to induce a controllable level of oxidation on a range of intact proteins. These experiments utilize a relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber, along with a unique cell activation approach to improve control over the intact protein oxidation yield. Studies were conducted to evaluate the level of protein adsorption onto the electrode surface. This report demonstrates a robust protocol for the use of BDD electrochemistry and high performance LC-MS/MS as a high-throughput experimental pipeline for probing higher order protein structure, and illustrates how it is complementary to predictive computational modeling efforts.