Fig. 1.

4-OI alkylates cysteine 22 of GAPDH. a The orthogonal projection to latent structures discriminant analysis (OPLS-DA) score plots compared control (CON) and LPS-stimulated (LPS) RAW264.7 macrophages (24 h). Results are from seven independent experiments. b Metabolite levels in CON versus LPS-induced RAW264.7 macrophages. Blue and red dots represent metabolites significantly downregulated and upregulated after LPS stimulation, respectively. c Representative LC-MS/MS spectra showing covalent modification of the cysteine 22-containing GAPDH peptide by 4-OI (+242.15 Da) after 4-OI treatment for 4 h in GAPDH immunoprecipitated from RAW264.7 macrophages. d Representative LC-MS/MS spectra showing covalent modification of the cysteine 22-containing GAPDH peptide by itaconate (+130.02 Da) after LPS stimulation for 6 h in GAPDH immunoprecipitated from RAW264.7 macrophages. e Representative LC-MS/MS spectra showing covalent modification of the cysteine 22-containing GAPDH peptide by 4-OI (+242.15 Da) in recombinant mouse GAPDH treated with 4-OI (500 μM) for 4 h at 37 °C. Da daltons. f Schematic diagram of GAPDH thiol group was covalent modified by 4-OI