title: S-Glutathionyl quantification in the attomole range using glutaredoxin-3-catalyzed cysteine derivatization and capillary gel electrophoresis with laser-induced fluorescence detection.
abstract: S-glutathionylation (Pr-SSG) is a specific post-translational modification of cysteine residues by the addition of glutathione. S-Glutathionylated proteins induced by oxidative or nitrosative stress play an essential role in understanding the pathogenesis of the aging and age-related disorder, such as Alzheimer's disease (AD). The purpose of this research is to develop a novel and ultrasensitive method to accurately and rapidly quantify the Pr-SSG by using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). The derivatization method is based on the specific reduction of protein-bound S-glutathionylation with glutaredoxin (Grx) and labeling with thiol-reactive fluorescent dye (Dylight 488 maleimide). The experiments were performed by coupling the derivatization method with CGE-LIF to study electrophoretic profiling in in vitro oxidative stress model-S-glutathionylated bovine serum albumin (BSA-SSG), oxidant-induced human colon adenocarcinoma (HT-29) cells, brain tissues, and whole blood samples from an AD transgenic (Tg) mouse model. The results showed almost an eightfold increase in S-glutathionyl abundance when subjecting HT-29 cells in an oxidant environment, resulting in Pr-SSG at 232 ± 10.64 (average ±SD; n=3) nmol/mg. In the AD-Tg mouse model, an initial quantitative measurement demonstrated the extent of protein S-glutathionylation in three brain regions (hippocampus, cerebellum, and cerebrum), ranging from 1 to 10 nmol/mg. Additionally, we described our developed method to potentially serve as a highly desirable diagnostic tool for monitoring S-glutathionylated protein profile in minuscule amount of whole blood. The whole blood samples for S-glutathionyl expression of 5-month-old AD-Tg mice are quantified as 16.3 μmol/L (=7.2 nmol/mg protein). Altogether, this is a fast, easy, and accurate method, reaching the lowest limit of Pr-SSG detection at 1.8 attomole (amol) level, reported to date.
authors.affiliation: Department of Biomedical Engineering, Louisiana Tech University, 818 Nelson Avenue, Ruston, LA 71272, USA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.forename: Cynthia
authors.initials: C
authors.lastname: Rodriguez
authors.forename: Magdalena L
authors.initials: ML
authors.lastname: Circu
authors.forename: Tak Yee
authors.initials: TY
authors.lastname: Aw
authors.forename: June
authors.initials: J
authors.lastname: Feng
mesh_terms: D000544:Alzheimer Disease / Q000175:diagnosis; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D004195:Disease Models, Animal; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D054477:Glutaredoxins / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D007834:Lasers; D051379:Mice; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D054477:Glutaredoxins; D012710:Serum Albumin, Bovine; D005978:Glutathione; D003545:Cysteine