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 good 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.

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

“…Peptides are mostly derivatized with fluorescent labels to become detectable with LIF detectors, the sensititivity of which is about two to three orders higher than that of the most frequently used and the derivatization‐free UV‐spectrophotometric absorbance detectors. A new fluorescent label, 1,3,5,7‐tetramethyl‐8‐phenyl‐(2‐maleimide)‐difluoroboradiaza‐ s ‐indacene, providing low background, high sensitivity, and fast reactivity, was used for derivatization of glutathione (GSH) and other thiol compounds . After precolumn derivatization in sodium phosphate buffer, pH 7.40, at 37°C for 6 min, GSH and three other thiols (cystein, homocysteine, γ‐glutamylcysteine) were separated by CZE in BGE composed of 10 mM sodium citrate, pH 7.5, within 11 min.…”

Design and Application of Sensors for Chemical Cytometry

“…Fluorescence detection is typically performed on-column by interrogating the capillary with a focused light source, generally a laser, and detecting emitted light with a photomultiplier tube (PMT) or photodiode. 18, 21, 26, 45 ED coupled to CE offers a multitude of advantages for single cell analysis due to its high sensitivity, low LOD, and low cost. 23, 25, 49, 50 MS coupled to CE has led to impressive advances in elucidation of the proteome and metabolome of single cells.…”

Capillary electrophoresis in the analysis of biologically important thiols

“…Thiol content (GSH, Cys, Hcy, and ϒ‐GluCys) in a single cell was analyzed by Guo et al. with chemical cytometry, a postcolumn sheet flow cuvette attached to a CE separation capillary for fluorescence measurement. The content of GSH and Cys in two types of cancer cells (HCT‐29 and MCF‐10A) was analyzed with LODs as low as 13–21 pM.…”

“…Human umbilical vein endothelial cells (ECV304) and R1 stem cells were analyzed for thiol content by sensitive CE method with LIF detection with nanomolar concentration sensitivity. Other researchers have analyzed the content of Caucasian colon adenocarcinoma grade II cell line (HT‐29) , preneoplastic mammary epithelial cells (MCF‐10A) , human breast cancer cells MDA‐MB‐231 , SMMC‐7221 apoptotic cells , and RAW264.7 cells . Wang et al.…”