title: In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells.
abstract: Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.
authors.affiliation: †Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46204, United States.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Espino
authors.affiliation: ‡Avon High School, Avon, Indiana 46123, United States.
authors.forename: Vishaal S
authors.initials: VS
authors.lastname: Mali
authors.affiliation: †Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46204, United States.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Jones
mesh_terms: D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002522:Chlorocebus aethiops; D006861:Hydrogen Peroxide / Q000737:chemistry; D013058:Mass Spectrometry; D023302:Protein Footprinting; D011506:Proteins / Q000737:chemistry*; D014709:Vero Cells
chemical_list: D011506:Proteins; D006861:Hydrogen Peroxide