Probing the Conformational Changes of a Model Protein by In-Cell Footprinting Coupled with Mass Spectrometry

Abstract

In recent years, protein footprinting coupled with mass spectrometry has been used to identify protein-protein interaction sites and regions of conformational change through modification of solvent accessible sites in proteins. Hydroxyl radical-based footprinting (HRBF) approaches utilize hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. One HRBF method, fast photochemical oxidation of proteins (FPOP), utilizes an excimer laser for photolysis of hydrogen peroxide to generate hydroxyl radicals. To date, HRBF methods have been used in vitro on relatively pure protein systems. We have further extended the FPOP method for in cell analysis of proteins. This will allow for study of proteins in their native cellular environment and be especially useful for the study of membrane proteins which can be difficult to purify for in vitro studies. We have designed and built a single cell flow system to enable uniform access of cells to the laser. Results demonstrate that in cell FPOP (IC-FPOP) can oxidatively modify over 1300 proteins in various cellular compartments. We have recently tested the ability of IC-FPOP to detect conformational changes in-cell using GCaMP2 as a model protein. GCaMP2, a chimeric protein of GFP and calmodulin, undergoes a conformational change upon calcium binding. IC-FPOP successfully identifies regions of conformational change similar to in vitro FPOP. This represents the first time in-cell footprinting coupled with mass spectrometry has be used to probe protein structure in-cell.