Figure 2.

The ²⁷²CXXC²⁷⁵ motif in CBS protein exists in disulfide and thiol states under oxidative and reductive conditions. A, recombinant CBS protein was analyzed by LC-MS/MS. The mass spectrum of the KCPGCRIIGVDPE peptide that contained the oxidized form of the ²⁷²CXXC²⁷⁵ motif is shown. The monoisotopic mass [M+2H]²⁺ of this peptide was 692.8389 Da (observed nominal mass = 1384.6711 Da; expected nominal mass = 1384.6712 Da). The chromatographic peaks of the oxidized peptide from untreated CBS and reduced CBS are shown in the inset. B, recombinant CBS was treated with 10 mm DTT for 1 h and then was analyzed by LC-MS/MS. The mass spectrum of the KCPGCRIIGVDPE peptide showing the reduced form of ²⁷²CXXC²⁷⁵ motif is shown. The monoisotopic mass [M+2H]²⁺ of this iodoacetamide-derivatized peptide was 750.8683 Da (observed nominal mass = 1500.7297 Da; expected nominal mass = 1500.7297 Da). The chromatographic peaks of the reduced peptide from the untreated CBS and reduced CBS are shown in the inset. C, the recombinant CBS was incubated without or with 10 mm DTT for 1 h and then precipitated with trichloroacetic acid. The precipitated CBS was labeled with the methyl-PEG-maleimide reagent MM(PEG)₂₄. The labeled samples were resolved on SDS-PAGE and immunoblotted for CBS. The molecular size markers in kDa are indicated at the left.