dc.contributor.author | Albertolle, Matthew E. | |
dc.contributor.author | Glass, Sarah M. | |
dc.contributor.author | Trefts, Elijah | |
dc.contributor.author | Guengerich, F. Peter | |
dc.date.accessioned | 2020-04-28T19:44:23Z | |
dc.date.available | 2020-04-28T19:44:23Z | |
dc.date.issued | 2019-04-19 | |
dc.identifier.citation | Albertolle, M. E., Glass, S. M., Trefts, E., & Guengerich, F. P. (2019). Isotopic tagging of oxidized and reduced cysteines (iTORC) for detecting and quantifying sulfenic acids, disulfides, and free thiols in cells. The Journal of biological chemistry, 294(16), 6522–6530. https://doi.org/10.1074/jbc.AC118.007225 | en_US |
dc.identifier.other | eISSN: 1083-351X | |
dc.identifier.uri | http://hdl.handle.net/1803/9973 | |
dc.description.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 x 10(5) m(-1) s(-1) (pH 8.0, 23 degrees 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 1-1 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. | en_US |
dc.description.sponsorship | This work was supported, in whole or in part, by National Institutes of Health Grants R01 GM118122 (to F. P. G.), F31 HL136133 (to M. E. A.), T32 ES007028 (to F. P. G. and S. M. G.), F31 DK112553 (to E. T.), R37 DK050277 (to E. T.), and R01 DK054902 (to E. T.) and American Heart Association Pre-doctoral Fellowship PRE33410007 (to M. E. A.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. | en_US |
dc.language.iso | en | en_US |
dc.publisher | Journal of Biological Chemistry | en_US |
dc.subject | thiol | en_US |
dc.subject | disulfide | en_US |
dc.subject | mass spectrometry (MS) | en_US |
dc.subject | proteomics | en_US |
dc.subject | oxidative stress | en_US |
dc.subject | oxidation-reduction (redox) | en_US |
dc.subject | cysteine | en_US |
dc.subject | isotopic tagging of oxidized and reduced cysteines (iTORC) | en_US |
dc.subject | oxidative damage | en_US |
dc.subject | sulfenic acid | en_US |
dc.title | Isotopic tagging of oxidized and reduced cysteines (iTORC) for detecting and quantifying sulfenic acids, disulfides, and free thiols in cells | en_US |
dc.type | Article | en_US |