Isotopic tagging of oxidized and reduced cysteines (iTORC) for detecting and quantifying sulfenic acids, disulfides, and free thiols in cells.

ABSTRACT: Oxidative modifications of cysteine residues are an important component in signaling pathways, enzymatic regulation, and redox homeostasis. Current direct and indirect methods detect specific modifications and a general binary population of "free" or "oxidized" cysteines, respectively. In an effort to combine both direct and indirect detection strategies, here we developed a method that we designate isotopic tagging of oxidized and reduced cysteines (iTORC). This method uses synthetic molecules for rapid isotopic coding of sulfenic acids, reduced cysteines, and disulfides in cells. Our approach utilizes isotopically distinct benzothiazine and halogenated benzothiazine probes to sequentially alkylate sulfenic acids and then free thiols and, finally, after a reduction step, cysteines oxidized to disulfides or other phosphine-reducible states. We ascertained that the iodinated benzothiazine probe has reduced cross-reactivity toward primary amines and is highly reactive with the cysteine of GSH, with a calculated rate constant of 2 × 10⁵ m^-1 s^-1 (pH 8.0, 23 °C) (i.e. 10-20 times faster than N-ethylmaleimide). We applied iTORC to a mouse hepatocyte lysate to identify known sulfenylated and disulfide-bonded proteins, including elongation factor 1-α1 and mouse serum albumin, and found that iTORC reliably detected their expected oxidation status. This method can be easily employed to study the effects of oxidants on recombinant proteins and cell and tissue extracts, and the efficiencies of the alkylating agents enable completion of all three labeling steps within 2 h. In summary, we demonstrate here that halogenated benzothiazine-based alkylating agents can be utilized to rapidly measure the cellular thiol status in cells.

SUBMITTER: Albertolle ME 

PROVIDER: S-EPMC6484142 | biostudies-literature |

REPOSITORIES: biostudies-literature