With the norbornene probes validated on a protein model, cell lysates were examined next. To determine optimal labeling conditions, the concentration of H 2 O 2 and probes 1 and 2 were systematically varied (S15-S20). Accordingly, HeLa cell lysates were treated with the norbornene probe (0.1 to 3 mM), followed by H 2 O 2 (0 to 2 mM) for 1 h at room temperature. Samples were then purified with size exclusion columns to remove excess probe. For probe 1, samples were analyzed directly by SDS-PAGE and western blotting. For probe 2, samples were ligated to an azide-tagged biotin derivative prior to SDS-PAGE. Labeling profiles were also compared to the widely used dimedone derivative DCP-Bio1 (6) using the same protocol (Fig. ). For both norbornene probes, increased labeling was observed with increased probe concentration and increased H 2 O 2 concentration (Fig. ). As a control, lysates were also pre-treated with H 2 O 2 (2 mM) for 30 minutes before addition of the norbornene probes. In this case, no significant protein labeling was observed. Overoxidation to the sulfinic or sulfonic acids is likely, neither of which react with the norbornene-based probes 7b (Fig. , far right lane). When the norbornene probes were added to lysate pre-treated with the reducing agent tris(2carboxyethyl)phosphine (TCEP), no labeling was observed because any endogenous sulfenic acid would be reduced by TCEP (Fig , far left lane). This outcome also indicates that the norbornene probe does not react with cellular thiols under the conditions of the assay. Together, the results in Figure indicate that norbornene probes are highly selective for cysteine sulfenic acid, even in a complex protein mixture. Non-selective labeling was only observed if excess 1 was not removed before denaturing proteins at elevated temperatures (~95 ºC, S22). Under these conditions it is possible that the norbornene probes can react directly with thiols through a thiol-ene reaction. Therefore, to avoid these false positives, it is important to remove excess probe with a desalting column or sizeexclusion chromatography before denaturing proteins for SDS-PAGE analysis.