Fig. 3: Glutathione-associated isotopologue similarity subnetwork reveals previously unknown metabolic reactions.

a The glutathione-associated isotopologue similarity subnetwork in positive ionization mode. The grey shad highlights the edge between glutathione and M292T447. b The unknown metabolic reaction between glutathione and M292T447. Orange points represent the ¹³C-labeled atoms. c Elucidation of the metabolic reaction between glutathione and M292T447 (γ-Glu-Ser-Gly). The red shads highlight the variable substructures. d Interpretation of MS/MS spectra between glutathione and γ-Glu-Ser-Gly. The red shads highlight the variable substructures. e Validation of γ-Glu-Ser-Gly using chemical standard (Std): retention time match (top) and MS2 spectral match (bottom). f The relative levels of γ-Glu-Ser-Gly in various mouse tissues (n = 3–4) quantified by LC − MS. g Isotopologue pattern similarity between glutathione and γ-Glu-Ser-Gly in mouse liver tissue. Mice were administered with [U-¹³C]-glucose for 24 h (n = 4 biological replicates per group). h Intracellular γ-Glu-Ser-Gly levels in 293T and iBMDM cells after the treatment of buthionine sulfoximine (BSO; 100 μM) for 12 h or 10 h, respectively (n = 6 biological replicates for 293T cells per group, p = 0.0070 for glutathione, p < 0.0001 for γ-Glu-Ser-Gly; n = 5 biological replicates for iBMDM cells per group p = 0.0093 for glutathione, p < 0.0001 for γ-Glu-Ser-Gly). i Quantitative real-time PCR analyses of mRNA levels of GCS gene expression in control group and the siRNA-transfected 293T cells (n = 3 biological replicates per group; p = 0.023, ctrl vs siRNA1; p = 0.025, ctrl vs siRNA2; p = 0.031, ctrl vs siRNA3). j, k Intracellular levels of glutathione (p = 0.000064, ctrl vs siRNA1; p = 0.0019, ctrl vs siRNA2; p = 0.0015, ctrl vs siRNA3) ( j) and γ-Glu-Ser-Gly (p = 0.021, ctrl vs siRNA1; p = 0.011, ctrl vs siRNA2; p = 0.015, ctrl vs siRNA3) (k) in control group and siRNA-transfected 293T cells (n = 3 biological replicates per group). P-values were determined by a two-tailed Student’s t-test. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. Values represent means ± SD. Source data are provided as Source Data files for Fig. 3c and f–k.