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.

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.