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. Author manuscript; available in PMC: 2017 Jun 23.
Published in final edited form as: Cell Chem Biol. 2016 Jun 9;23(6):727–737. doi: 10.1016/j.chembiol.2016.05.008

Figure 3. Concentration-dependent analysis of cysteine-reactivity upon GSNO treatment.

Figure 3

(A) The light-tagged proteomes were treated with 0, 50, 100, 200, or 500 µM GSNO for 1 hr and compared to a buffer-treated heavy-tagged sample. Heavy:light ratio values (R) obtained for each identified cysteine at every GSNO concentration are shown in Table S2. Representative extracted ion chromatograms (light:red; heavy:blue) are shown for a cysteine that is insensitive to GSNO (GSTO1 Cys32) and two cysteines with high GSNO sensitivity (CTSD Cys329 and HADH2 Cys58). Cysteines with high sensitivity to GSNO show a clear concentration-dependent increase in R-values. (B) R-values are plotted with increasing GSNO concentration for six cysteines (from CLIC4, GSTO1, DCXR, HADH2, CTSD, and GAPDH) with varying GSNO sensitivities. The quantitative nature of the analysis allows ranking of cysteines by their GSNO sensitivity. (C) Corroboration of the MS-based identification of cysteine sensitivity and insensitivity using a gel-based assay for two proteins. Myc/His-tagged GSTO1 or CTSD overexpressing cell lysates were treated with varying GSNO concentrations followed by IA labeling. IA-labeled proteins were tagged with biotin using CuAAC and subjected to streptavidin enrichment, SDS-PAGE analysis, and immunoblotting with an anti-Myc antibody. A decrease in CTSD enrichment was observed at increasing GSNO concentrations, indicative of decreased cysteine reactivity and therefore IA labeling upon GSNO treatment. In contrast, no change in IA labeling and GSTO1 enrichment was observed at increasing GSNO concentrations, indicating insensitivity of the GSTO1 cysteines to GSNO.