Figure 4.

ESI-MS spectra of oxidized and S-glutathionylated GALDH. A, ESI-MS spectra of oxidized GALDH. Shown are mass spectra of untreated wild-type GALDH (black), GALDH incubated with hydrogen peroxide for 1 h (red), and GALDH incubated with hydrogen peroxide for 3 h (blue). Spectra were recorded under denaturing conditions in 50 mm ammonium acetate solution with 5% formic acid. The inset shows a zoom in of the overlayed mass spectra at charge state 54+. Under denaturing conditions FAD dissociates from GALDH, resulting in apo-GALDH and FAD in the mass spectra. Singly charged FAD is observed via protonation, and cationization by Na⁺ and K⁺. B, ESI-MS spectra of S-glutathionylated GALDH. Shown are mass spectrum of GALDH incubated with hydrogen peroxide and GSH (black) and mass spectrum of S-glutathionylated GALDH treated with DTT (red). Both spectra only show a zoom in on the charge states 44+ to 46+. Spectra were recorded under denaturing conditions in 50 mm ammonium acetate solution with 5% formic acid.