Chemical cytometry of thiols using capillary zone electrophoresis-laser induced fluorescence and TMPAB-o-M, an improved fluorogenic reagent.
Chemical cytometry of thiols using capillary zone electrophoresis-laser induced fluorescence and TMPAB-o-M, an improved fluorogenic reagent.
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DOI：
10.1039/c5an02116b
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发表时间：
2016-02-21
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影响因子：
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通讯作者：
Dovichi NJ
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作者：
Guo XF;Arceo J;Huge BJ;Ludwig KR;Dovichi NJ
低分子量硫醇化合物在许多生理过程中起着至关重要的作用。大多数测定硫醇化合物的方法是种群平均的；单细胞中硫醇类化合物的定量方法很少。本文报道了一种利用性能良好的荧光探针- 1,3,5,7-四甲基-8-苯基-（2-马来酰亚胺）-二氟硼砂-s-indacene (TMPAB-o-M)，结合毛细管区带电泳和柱后鞘流试管激光诱导荧光检测，测定单细胞中硫醇类化合物的超灵敏方法。TMPAB-o-M具有低本底、高灵敏度和优异的反应性。对分离方法进行优化后，在11 min内实现了标记谷胱甘肽（GSH）、半胱氨酸（Cys）、同型半胱氨酸和γ-谷氨酰半胱氨酸的基线分离，检测限为10 ~ 20 pM，质量lod为65 ~ 100 zmol。该方法适用于细胞匀浆和单个HCT-29和MCF-10A细胞中含硫醇化合物的分析。GSH是主要的硫醇，在两种细胞类型中也检测到Cys。细胞用n -乙基马来酰亚胺处理，显著降低了硫醇水平。
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.