Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes, as well as the formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively ‘tagging’ them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures, as selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamido cyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer) and biotin. We loaded biotin dimedone (0.1mM, 30min) into rat ventricular myocytes, treated them with H2O2 (0.1-10,000M, 5min) and monitored derivatization on Westerns using streptavidin-HRP. There was a dose-dependent increase in labeling of multiple proteins, which was maximal at 0.1 or 1mM H2O2, declining sharply below basal with 10mM treatment. Cell wide labeling was observed in fixed cells, probed with avidin-FITC using a confocal fluorescence microscope. Similar H2O2-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was re-supplied; highlighting the protein sulfenates as oxygen sensors. Cardiac proteins which were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with online analysis by mass spectrometry.