Fig. 6: GSK3 inhibition selectively perturbs the S-sulfenome.

a Workflow for quantitative S-sulfenome analysis. HeLa cells were treated with inhibitor (1 µM, 1 h) or vehicle as indicated. Lysates were prepared and treated with BTD (5 mM, 1 h) to label sulfenic acids. BTD-labeled proteomes with or without inhibitor treatment were digested and conjugated to heavy or light azide-tagged photocleavable biotin, respectively. Light and heavy labeled samples then were combined, clicked to biotin, enriched on streptavidin resin, and analyzed by LC-MS/MS. A heavy to light ratio (R_H/L) calculated for each BTD-labeled cysteine residue reports its relative level in inhibitor-treated samples versus controls. b Box plot showing a side-by-side comparison of results from GSK3 inhibitors Bio, BIM-IX, and SB216763. from two biological replicates. c Volcano plots showing the log2 values of the ratio between GSK3 inhibitor (heavy) and control (light) channels and the -log10(P) of the statistical significance in a two-sample t-test for all quantified cysteines. Sulfenation events that were significantly changed with GSK3 inhibition are shown in red (R_H/L > 1.5, P < 0.01). d Representative extracted ion chromatograms (XICs) showing changes in BTD-labeled peptides from the proteins as indicated. The profiles for light- and heavy-labeled peptide are shown in red and blue, respectively. The average R_H/L values calculated from two biological replicates are displayed below each individual XIC. e GO enrichment of the sub-sulfenome perturbed by GSK3 inhibition.