title: Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Louisville, Louisville, KY, 40292, USA.
authors.forename: Sadakatali S
authors.initials: SS
authors.lastname: Gori
authors.forename: Pawel
authors.initials: P
authors.lastname: Lorkiewicz
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Ehringer
authors.forename: Alex C
authors.initials: AC
authors.lastname: Belshoff
authors.forename: Richard M
authors.initials: RM
authors.lastname: Higashi
authors.forename: Teresa W-M
authors.initials: TW
authors.lastname: Fan
authors.forename: Michael H
authors.initials: MH
authors.lastname: Nantz
mesh_terms: D045744:Cell Line, Tumor; D003545:Cysteine; D004151:Dipeptides; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D015335:Molecular Probes / Q000138:chemical synthesis; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D015335:Molecular Probes; D013438:Sulfhydryl Compounds; C028505:cysteinylglycine; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide; D007460:Iodoacetamide