title: Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry.
abstract: Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically requires a high concentration (>200 mM) of the chemical reducing agent Tris(2-carboxyethyl)phosphine (TCEP) as its reduction rate constant is decreased at low pH and temperature. Serious adverse effects on chromatographic and mass spectrometric performances have been reported when using high concentrations of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, Odense M DK-5230, Denmark.
authors.forename: Simon
authors.initials: S
authors.lastname: Mysling
authors.forename: Rune
authors.initials: R
authors.lastname: Salbo
authors.forename: Michael
authors.initials: M
authors.lastname: Ploug
authors.forename: Thomas J D
authors.initials: TJ
authors.lastname: Jørgensen
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D011487:Protein Conformation; D055293:Receptors, Urokinase Plasminogen Activator / Q000737:chemistry*
chemical_list: D004220:Disulfides; D055293:Receptors, Urokinase Plasminogen Activator