title: Chemical cytometry of thiols using capillary zone electrophoresis-laser induced fluorescence and TMPAB-o-M, an improved fluorogenic reagent.
abstract: Low molecular weight thiol compounds play crucial roles in many physiological processes. Most methods for determination of thiol compounds are population-averaged; few methods for quantification of thiol compounds in single cells have been reported. We report an ultrasensitive method for determination of thiol compounds in single cells by use of 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), a fluorogenic probe with useful spectral properties, coupled with capillary zone electrophoresis and laser induced fluorescence detection using a post-column sheath flow cuvette. TMPAB-o-M provides low background, high sensitivity, and excellent reactivity. After optimization of the separation method, we achieved baseline separation of labeled glutathione (GSH), cysteine (Cys), homocysteine, and γ-glutamylcysteine within 11 min, and produced concentration limits of detection from 10 to 20 pM and mass LODs of 65 to 100 zmol. The method was applied for analysis of thiol containing compounds in both cell homogenates and in single HCT-29 and MCF-10A cells. GSH was the main thiol, and Cys was also detected in both cell types. Cells were treated with N-ethylmaleimide, which significantly attenuated thiol levels.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. ndovichi@nd.edu and Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China.
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Guo
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. ndovichi@nd.edu.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Arceo
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. ndovichi@nd.edu.
authors.forename: Bonnie Jaskowski
authors.initials: BJ
authors.lastname: Huge
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. ndovichi@nd.edu.
authors.forename: Katelyn R
authors.initials: KR
authors.lastname: Ludwig
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. ndovichi@nd.edu.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry*; D006801:Humans; D007834:Lasers; D057230:Limit of Detection; D008301:Maleimides / Q000737:chemistry*; D017785:Photons; D059010:Single-Cell Analysis / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: C534661:1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D008301:Maleimides; D013438:Sulfhydryl Compounds
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