We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue*QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4±11.5 and 10.1±4.0 nmol/mg protein, respectively.
|Number of pages||9|
|Journal||Analytical and Bioanalytical Chemistry|
|State||Published - Jul 2014|
Bibliographical noteFunding Information:
Acknowledgments This work was supported by grants from NIH (1 R01 ES022191-01, 3R01CA118434-02S1, and 1R01CA118434-01A2). The FT-ICR-MS instrumentation at the Center for Regulatory and Environmental Analytical Metabolomics mass spectrometry facility was funded by an NSF/EPSCoR grant (EPS-0447479). We also acknowledge the insightful contributions on data analysis from Mr. James Carrier and Dr. Hunter Moseley (Department of Molecular and Cellular Biochemistry, University of Kentucky), and we thank Ms. Stephanie Mattingly for her advice on how to best operate the FT-ICR-MS for isotopologue pairs of quaternary ammonium derivatives. We also thank Ms. Ramya Balasubramaniam for providing cultured cells.
- Oxidative stress
ASJC Scopus subject areas
- Analytical Chemistry