title: Mapping pH-induced protein structural changes under equilibrium conditions by pulsed oxidative labeling and mass spectrometry.
abstract: Mass spectrometry (MS)-based protein conformational studies are a rapidly growing field. The characterization of partially disordered conformers is of particular interest because these species are not amenable to classical high-resolution techniques. Such equilibrium intermediates can often be populated by exposure to mildly acidic pH. Hydroxyl radical (·OH) introduces oxidative modifications at solvent-accessible side chains, while buried sites are protected. ·OH can be generated by laser photolysis of H(2)O(2) (fast photochemical oxidation of proteins-FPOP). The resulting labeling pattern can be analyzed by MS. The characterization of partially disordered intermediates usually involves comparative measurements under different solvent conditions. It can be challenging to separate structurally induced labeling changes from pH-mediated "secondary" effects. The issue of secondary effects in FPOP has received little prior attention. We demonstrate that with a proper choice of conditions (e.g., in the absence of pH-dependent ·OH scavengers) such undesired phenomena can be almost completely eliminated. Using apomyoglobin as a model system, we map the structure of an intermediate that is formed at pH 4. This species retains a highly protected helix G that is surrounded by partially protected helices A, B, and H. Our results demonstrate the utility of FPOP for the structural characterization of equilibrium intermediates. The near absence of an intrinsic pH dependence represents an advantage compared to hydrogen/deuterium exchange MS.
authors.affiliation: Department of Chemistry, Western University, London, Ontario, N6A 5B7, Canada.
authors.forename: Siavash
authors.initials: S
authors.lastname: Vahidi
authors.forename: Bradley B
authors.initials: BB
authors.lastname: Stocks
authors.forename: Yalda
authors.initials: Y
authors.lastname: Liaghati-Mobarhan
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D017665:Hydroxyl Radical / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry*; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D001059:Apoproteins; D009211:Myoglobin; D012997:Solvents; C010857:apomyoglobin; D017665:Hydroxyl Radical