Interestingly, dimedone probe 6 led to a different labeling profile than norbornene probes 1 and 2 (Fig. ). The different protein hits may reflect the different size and distinct reaction mechanisms of the norbornene and dimedone probes. Dimedone probe 6 also labeled proteins in the control experiment in which the lysates were pretreated with H 2 O 2 30 minutes before the addition of the probe (Fig. , far right lane). It was thought that under these conditions cysteine sulfenic acids would be overoxidized before the addition of the probe, so 6 may react unselectively in this situation. It is known, for instance, that dimedone can react with amines, even at room temperature under certain conditions, but it is not clear what is causing the non-selective labeling in this experiment. Non-selective labeling was also observed when 6 was added to cell lysates pre-treated with the reducing agent TCEP (Fig. , far left lane). Previous studies have indicated that 6 can label proteins nonselectively if cysteine thiols are not alkylated. The presence of TCEP apparently exacerbates this problem by increasing thiol concentration through reduction of protein disulfides. A similar outcome was observed if DTT was used in the pre-reduction-again leading to non-selective labelling by dimedone derivative 6 (S23-S24). Even with attempts to remove excess 6 before SDS-PAGE analysis, off-target labelling was observed (S23-S24). These results indicate dimedone probe 6 is not as selective as norbornene derivatives 1 and 2. These results also prompt a cautionary note that false positives are possible when using probe 6 in the presence of free thiols or in experiments in which reducing conditions are established by adding TCEP or DTT to protein mixtures.