Fig. 3: Defining the oxidation-sensitive C. elegans redoxome.
a Schematic diagram of our quantitative chemoproteomic workflow for profiling cysteine redox forms in C. elegans upon peroxide treatment. Worms treated with (red) or without (blue) H2O2 were labeled in vitro with the chemoselective probes IPM (for –SH), BTD (for –SOH), or DiaAlk (for –SO2H), in parallel (Fig. 1). The probe-tagged proteomes were processed into tryptic peptides, followed by reactions with light or heavy Az-UV-biotin reagents via CuAAC. The light and heavy-labeled samples were then mixed equally in amount, cleaned with SCX, and enriched on streptavidin beads. Labeled peptides were then photoreleased and subjected to LC-MS/MS-based proteomic analysis for identification and quantification of individual cysteine residues. b Distribution of the Log-transformed RT/C values for cysteine redox forms. c Heatmap showing dynamic changes in redox forms for cysteine sites identified by all three probes. d, e Representative XICs showing changes in probe-labeled peptides from Y41D4A.5 (C120) (d) and GPD-3 (C158) (e). Profiles for light- and heavy-labeled peptides are shown in red and blue, respectively. The average RT/C values calculated from two biological replicates are displayed below each XIC. f Structure of GPD-3 ortholog showing the distance between C158 and C162. g A Pie chart showing the distribution of the number of dynamically-changed cysteine redox forms per protein in the C. elegans redoxome. h A selected group of known redox-reactive metabolic enzymes identified in this study. Functionality information is retrieved from the UniProt knowledge base.
