title: Protein-bound glutathione measurement in cultured cells by CZE with LIF detection.
abstract: Protein modification due to S-glutathio(ny)lation, usually a reversible process in intact cells, arises interest as a possible mode of regulatory events that may potentially modify a large number of cellular processes. However, since less than 1% of the total protein is S-thiolated in resting cells, high sensitivity methods are required for its evaluation. We set up a new method by CE with LIF detection that allows to measure all forms of intracellular GSH involved in the process. For total and reduced glutathione, cell lysates were rapidly derivatized by 5-iodoacetoamidofluorescein (5-IAF), a selective reagent which traps thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 47 cmx75 microm id capillary by using 7 mmol/L sodium phosphate at pH 11.6. For the evaluation of S-glutathio(ny)lation, intracellular proteins from cell lysates were precipitated and washed to eliminate free GSH. After protein resuspension with NaOH and reduction treatment with tri-n-butylphosphine (TBP), the freed GSH was dried in a vacuum concentrator and directly dissolved in the derivatization mixture. GSH-IAF adduct was detected in a 6 mmol/L sodium phosphate, 3 mmol/L boric acid, and 75 mmol/L N-methylglucamine run buffer in less than 5 min. The high sensitivity ensured by 5-IAF use and sample concentration, allowed to quantify GSH at levels as low as 5 nmol/L, value suitable for the evaluation of protein S-glutathio(ny)lation. The method suitability was checked both in HUVEC and ECV304 cultured cells.
authors.affiliation: Department of Biomedical Sciences, University of Sassari, Sassari, Italy. angelozinellu@libero.it
authors.forename: Angelo
authors.initials: A
authors.lastname: Zinellu
authors.forename: Salvatore
authors.initials: S
authors.lastname: Sotgia
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Posadino
authors.forename: Valeria
authors.initials: V
authors.lastname: Pasciu
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Zinellu
authors.forename: Maria Franca
authors.initials: MF
authors.lastname: Usai
authors.forename: Bastianina
authors.initials: B
authors.lastname: Scanu
authors.forename: Roberto
authors.initials: R
authors.lastname: Chessa
authors.forename: Leonardo
authors.initials: L
authors.lastname: Gaspa
authors.forename: Bruna
authors.initials: B
authors.lastname: Tadolini
authors.forename: Luca
authors.initials: L
authors.lastname: Deiana
authors.forename: Ciriaco
authors.initials: C
authors.lastname: Carru
mesh_terms: D002138:Calibration; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000379:methods*; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
chemical_list: D011506:Proteins; D005978:Glutathione