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Fluorescent and Affinity-Based Tools To Detect Cysteine Sulfenic Acid Formation in Proteins

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journal contribution
posted on 21.11.2007, 00:00 by Leslie B. Poole, Chananat Klomsiri, Sarah A. Knaggs, Cristina M. Furdui, Kimberly J. Nelson, Michael J. Thomas, Jacquelyn S. Fetrow, Larry W. Daniel, S. Bruce King
Cysteine sulfenic acid formation in proteins results from the oxidative modification of susceptible cysteine residues by hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This species represents a biologically significant modification occurring during oxidant signaling or oxidative stress, and it can modulate protein function. Most methods to identify such oxidatively modified proteins rely on monitoring the loss of one or more thiol group(s) or on selective labeling of nascent thiol groups following reduction of oxidized proteins. Our previous work reported the direct labeling of these chemically distinct modifications with a dimedone analogue, 1,3-cyclohexadione, to which a linker and functional group (an alcohol) had been added; further addition of a fluorescent isatoic acid or methoxycoumarin reporter allowed detection of the incorporated tag by fluorescence techniques (Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M., and King, S. B. (2005) Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjugate Chem. 16, 1624–1628). We have now expanded our arsenal of tagging reagents to include two fluorescein-, two rhodamine-, and three biotin-conjugated probes based on the original approach. The new tools provide readily detectable fluorescent and affinity probes to identify sulfenic acid modifications in proteins and have been used in subsequent mass spectrometric analyses to confirm covalent attachment of the conjugates and directly determine the site of modification.