The norbornene probes were tested in living cells next. The toxicity of probes 1 and 2, the DMSO vehicle, and H 2 O 2 were first assessed over a 5 hour exposure period to help identify conditions suitable for live cell analysis. The concentration of 1 was tolerated up to 3 mM, with 75-80% viability over 72 hours after replacing the growth media (S26). Probe 2, however, had limited solubility at 3 mM and was toxic to cells, leading to complete loss of cell viability (S26). Therefore, probe 2 was not considered further. The DMSO vehicle was tolerated up to 1% by volume, with 80% cell viability (S26). Generally, DMSO concentrations higher than 0.5% by volume can be toxic to cells. However, many live cell sulfenic acid studies have used DMSO concentrations up to 2%. H 2 O 2 was tolerated by cells up to 0.33 mM, but 1 mM H 2 O 2 led to total cell death (S26). Similar toxicity levels of H 2 O 2 have been reported previously, but the values vary widely. In these cases, cells are most likely dying due to extreme oxidative stress. Guided by this toxicity data, the labeling of cysteine sulfenic acid residues was tested in live cells (Fig. and S27-S29). In these experiments, HeLa cells were treated with a solution of 1 in DMSO so that the probe concentration was 1 or 3 mM and the final concentration of DMSO was 0.33% or 1% by volume, respectively. The cells were then incubated for 2 hours at 37 °C before the addition of H 2 O 2 . This initial incubation period was designed to allow the probe to enter the cell before inducing oxidative stress. After the addition of the H 2 O 2 , the cells were incubated for an additional 2 hours before they were harvested, washed, and lysed. The protein lysate was then analyzed by SDS-PAGE and western blotting. Protein labeling increased with increasing concentration of 1 and labeling was only observed in the presence of hydrogen peroxide (Fig. ). With increasing concentrations of H 2 O 2 , labeling also increased (S27-S28). In contrast to the cell lysate experiments in Figure , the same labeling was observed whether or not the protein mixtures were passed through a size exclusion column before SDS-PAGE and western blotting, so this precaution does not seem to be necessary for live cell experiments (S29). We are currently embarked on a dedicated proteomics study to annotate these hits and evaluate them against the known sulfenome.