Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan, Fukuoka 814-0180, Japan.
Anal Bioanal Chem. 2011 Feb;399(4):1665-75. doi: 10.1007/s00216-010-4461-6. Epub 2010 Dec 12.
In this paper we introduce a novel approach for highly selective and sensitive analysis of cysteines (glutathione, cysteine, and homocysteine). This method is based on the detection of intramolecular fluorescence resonance energy transfer (FRET) in a liquid chromatography (LC) system after double-labeling of the amino and sulfhydryl groups of the cysteines. In this detection process, we monitored the FRET between the amine-derivatized and thiol-derivatized fluorophores. We screened 16 combinations of fluorescent reagents. As a result, FRET occurred most effectively when the sulfhydryl and amino groups of the cysteines were derivatized with 7-diethylamino-3-[{4'-(iodoacetyl)amino}phenyl]-4-methylcoumarin (DCIA, Ex/Em 390/480 nm) and 4-fluoro-7-nitrobenz-2-oxo-1,3-diazole (NBD-F, Ex/Em 480/540 nm), respectively, in this order. The double-labeled cysteines emitted NBD-F fluorescence (540 nm) through an intramolecular FRET process when they were excited at the wavelength of maximum excitation of DCIA (390 nm). The generation of FRET was confirmed by comparison with analysis of n-amylamine or tryptophan (amines without a sulfhydryl group) and 6-mercaptohexanol (thiol without an amino group) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the double-labeled cysteines (DCIA and NBD-F) when performing LC on an ODS column with isocratic elution. The limits of quantification (signal-to-noise ratio = 10) and detection (signal-to-noise ratio = 3) for the cysteines, for a 20-μL injection volume, were in the range 150-670 fmol and 46-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of a system which takes advantage of conventional detection of the derivatives. Furthermore, this method provides sufficient selectivity and sensitivity to determine the total cysteines present in the plasma of healthy humans.
在本文中，我们介绍了一种用于高选择性和高灵敏度分析半胱氨酸（谷胱甘肽、半胱氨酸和同型半胱氨酸）的新方法。该方法基于在液相色谱（LC）系统中对半胱氨酸的氨基和巯基进行双重标记后，检测分子内荧光共振能量转移（FRET）。在这个检测过程中，我们监测了胺衍生化和硫醇衍生化荧光团之间的 FRET。我们筛选了 16 种荧光试剂的组合。结果表明，当半胱氨酸的巯基和氨基分别用 7-二乙氨基-3-[[4'-(碘乙酰基)氨基]苯基]-4-甲基香豆素（DCIA，Ex/Em 390/480nm）和 4-氟-7-硝基苯并-2-氧代-1,3-二唑（NBD-F，Ex/Em 480/540nm）衍生化时，FRET 最有效地发生。当用 DCIA 的最大激发波长（390nm）激发时，双标记的半胱氨酸通过分子内 FRET 过程发射 NBD-F 荧光（540nm）。通过与使用 LC 和三维荧光检测系统对半氨基己烷或色氨酸（无巯基的胺）和 6-巯基己醇（无氨基的硫醇）的分析进行比较，确认了 FRET 的产生。我们能够在 ODS 柱上通过等度洗脱进行 LC 分离双标记的半胱氨酸（DCIA 和 NBD-F）。对于 20μL 进样量，半胱氨酸的定量限（信噪比=10）和检测限（信噪比=3）分别为 150-670fmol 和 46-200fmol。分子内 FRET 形成衍生化方法的灵敏度高于利用常规衍生化衍生物检测的系统的灵敏度。此外，该方法提供了足够的选择性和灵敏度来确定健康人类血浆中存在的总半胱氨酸。
