In Cell Footprinting Coupled with Mass Spectrometry for the Structural Characterization of a Membrane Protein

Abstract

Footprinting methods coupled with mass spectrometry have been used in recent years to analyze protein-protein and protein-ligand interactions and regions of protein conformational change. The footprinting method fast photochemical oxidation of proteins (FPOP) utilizes hydroxyl radicals to oxidatively modify solvent accessible sites in proteins. We have extended the use of FPOP as an in cell method where we can directly probe the structure of proteins in their native cellular environment. In cell FPOP would be especially useful for the study of membrane proteins owing to the difficulty in purifying and solubilizing these proteins for in vitro studies. We have used in cell FPOP coupled with mass spectrometry to identify the lipid binding region of the adaptor protein angiomotin (Amot). Amot can control cell shape and migration through its membrane binding ability. Identifying the lipid binding regions of Amot will aid in understanding its role in membrane trafficking. By oxidatively modifying MC7 cells transfected with membrane bound Amot and cells transfected with the S175A Amot mutant, which is localized in the cytosol, we can determine the lipid binding region from the changes in solvent accessibility that occurs upon lipid binding. High resolution mass spectrometry is used to identify oxidatively modified residues and for quantitation of oxidation levels. This study demonstrates the efficacy of in cell FPOP as a method for analyzing membrane proteins.