Protein footprinting coupled with mass spectrometry has emerged in recent years as a valuable tool to study protein structure. One of these footprinting methods, hydroxyl radical footprinting (HRF), utilizes hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. The solvent accessibility of amino acids change upon ligand binding and protein conformational change and these changes are reflected in the oxidative modification pattern. There are multiple methods to generate hydroxyl radicals including Fenton chemistry and radiolysis of water. One method, fast photochemical oxidation of proteins (FPOP), utilizes an excimer laser for photolysis of hydrogen peroxide to generate hydroxyl radicals. FPOP has been successfully used to characterize protein-protein and protein-ligand interaction sites as well as regions of conformational change. We have extended the use of FPOP for analysis of proteins inside cells. This method, entitled in-cell FPOP (IC-FPOP), makes it possible to study proteins in their native cellular environment. The method probes solvent accessibility similar to <i>in vitro </i>FPOP demonstrating its potential for studying protein structure within the cell.

Hydroxyl radical footprinting coupled with mass spectrometry: Application for in-cell protein

2019 Spring National Meeting

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