Labeling of ALDH, and the CLIC protein family with carbon electrophile probes. (a), Labeling of WT, E269A, and C303A ALDH1 enzymes with the CA probe. (b),. Labeling of WT, E269A, and C303A ALDH1 enzymes with the SE probe. Top panel, fluorescent gel images shown in grayscale demonstrating the selective labeling of the E269A and C303A mutants with the CA and SE probes, respectively. Bottom panel, western blots confirming equivalent expression of WT, E269A and C303A ALDH1 enzymes using α-X-press antibodies. (c), Labeling of CLICs with the CA probe in lysates and in whole cells. Top panel, fluorescent gel images shown in grayscale. Bottom panel, western blots confirming expression of CLICs using α-myc antibodies. (d), Nitric oxide and oxidized glutathione treatment of CLICs. Lysates were treated with either 5 mM of the nitric oxide donor, diethylamine nitric oxide, sodium salt (DEANO) or 2 mM of oxidized glutathione (GSSG). Top panel, fluorescent gel images shown in grayscale. Bottom panel, western blots confirming expression of CLICs using α-myc antibodies.

Proteome and solution reactivities of carbon electrophile probes. (a), Panel of probes utilized in this study. (b), Proteome reactivity profiles for SE, CA and UK probes demonstrating the number of assigned peptides with unique labeling sites on nine nucleophilic amino acid residues; top panel, SE, middle panel, CA, bottom panel, UK. (c), Solution reactivity profiles for SE, CA and UK probes; top panel, SE, middle panel, CA, bottom panel, UK. Representative extracted ion chromatograms are shown for product formation upon reacting the probes with 20 equivalents of each amino acid derivative in PBS for 12 hrs.