title: Unequivocal determination of site-specific protein disulfide bond reduction potentials by top-down FTICR MS: characterization of the N- and C-terminal redox-active sites in human thioredoxin 1.
abstract: We report the reliable determination of equilibrium protein disulfide bond reduction potentials (E°') by isotope-coded cysteine alkylation coupled with top-down Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). This technique enables multiple redox-active sites to be characterized simultaneously and unambiguously without the need for proteolysis or site-directed mutagenesis. Our model system was E. coli thioredoxin, and we determined E°' for its CGPC active-site disulfide as -280 mV in accord with literature values. E°' for the homologous disulfide in human thioredoxin 1 (Trx1) was determined as -281 mV, a value considerably more negative than the previously reported -230 mV. We also observed S-glutathionylation of Trx1 and localized that redox modification to Cys72; E°' for the intermolecular disulfide was determined as -186 mV. Intriguingly, that value corresponds to the intracellular glutathione/glutathione disulfide (GSH/GSSG) potential at the redox boundary between cellular differentiation and apoptosis.
authors.affiliation: National High Magnetic Field Laboratory, Florida State University , 1800 E. Paul Dirac Drive, Tallahassee, Florida 32310, United States.
authors.forename: Jenna
authors.initials: J
authors.lastname: Scotcher
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Bythell
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
mesh_terms: D020134:Catalytic Domain; D017357:Cyclotrons; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D013879:Thioredoxins / Q000737:chemistry*
chemical_list: D004220:Disulfides; D013879:Thioredoxins