The protein was then analyzed by liquid chromatographymass spectrometry (LC-MS, Fig. ). Two clear signals were observed in the deconvoluted mass spectrum (Fig. ). The signal at 23619 Da is consistent with sulfoxide adduct 4, expected to form after reaction of the probe with the sulfenic acid of papain. This was the first demonstration of norbornene derivatives as cysteine sulfenic acid traps on proteins. The signal at 23457 Da is consistent with conversion of the active site cysteine to the sulfinic acid ( ), a product of over-oxidation (S5). Importantly, no reaction of 3 was observed with papain in the absence of H 2 O 2 (S6), demonstrating selective reaction of the norbornene probe with the sulfenic acid oxidation state of cysteine. Reaction at the active site cysteine of papain was consistent with enzyme activity assays (Fig. and). Treating papain (7 μM) with H 2 O 2 (2.8 mM) lead to oxidation and complete loss of enzyme activity (S10-S12). Treating the oxidized papain with reducing agent dithiothreitol (DTT), lead to partial recovery of activity (S10-S12). This result is consistent with the formation of cysteine sulfenic acid and higher oxidation states of cysteine and then reduction of the cysteine sulfenic acid back to the active thiol. In contrast, when norbornene derivative 3 was present during the oxidation at a concentration of 53 mM, no enzyme activity could be recovered using DTT because of the formation of the stable sulfoxide adduct 4 (Fig. ). Importantly, the activity of papain was not affected by 3 in the absence of H 2 O 2 (Fig. ), indicating it did not react with the active site thiol.