Characterization of the cysteine 106 HMGB1 oxidation status with the sera of APAP (530 mg/kg; 5 h) dosed mice. (A) Schematic overview of the location of cysteine 106 within the cytokine domain of murine HMGB1, which is characterized by MS/MS. (B) Nonreducing SDS-PAGE separation and Western analysis of reduced and oxidized HMGB1 isolated by immunoprecipitation from the sera of fed and fasted APAP-dosed mice (530 mg/kg; 5 h) and characterized by LC-MS/MS. HMGB1 was also isolated from Z-VAD.fmk pretreated APAP-dosed mice. (C) MS/MS analysis of oxidized murine HMGB1 peptide 97–112 from a fed APAP-treated mouse containing the cysteine 106 sulfonic acid. (D) MS/MS analysis of reduced murine HMGB1 peptide 97–112 containing the cysteine 106 thiol from an APAP-treated mouse that was prefasted for 24 h. MS/MS and Western figures are representative of six mice per group carried out in three independent investigations.