Dept. Biomedical Sciences, University of Sassari, Sassari, Italy.
J Sep Sci. 2010 Jan;33(1):126-31. doi: 10.1002/jssc.200900582.
We describe a new method for the quantification of low molecular weight thiols, as homocysteine, cysteine, cysteinylglycine, glutamylcysteine and glutathione bound to human plasma albumin. After albumin isolation and purification by SDS-PAGE, thiols were freed from protein with tri-n-butylphosphine and successively derivatized with 5-iodoacetamidofluorescein. Samples were then injected and quantified in about 18 min by CE with laser induced fluorescence detection. Precision tests indicate a good repeatability of the method both for migration times (RSD<0.63%) and areas (RSD<2.98%). The method allows to measure all five low molecular weight thiols released from just 3 microg of albumin thus improving the other described methods in which only three or four thiols were detected. Due to the elevated sensitivity (LOD of 0.3 pM for all thiols), also low molecular weight thiols bound to albumin filtered in tissues could be quantified.
我们描述了一种新的方法来定量分析与人体血浆白蛋白结合的低分子量硫醇，如同型半胱氨酸、半胱氨酸、半胱氨酰甘氨酸、谷氨酰半胱氨酸和谷胱甘肽。在 SDS-PAGE 分离和纯化白蛋白后，用三丁基膦将硫醇从蛋白质中释放出来，并依次用 5-碘乙酰胺荧光素进行衍生化。然后，通过 CE 进行注射，并通过激光诱导荧光检测在大约 18 分钟内定量。精密度测试表明，该方法对于迁移时间（RSD<0.63%）和面积（RSD<2.98%）均具有良好的重复性。该方法仅需 3μg 白蛋白即可释放并测量所有五种低分子量硫醇，从而改进了其他描述的方法，这些方法仅能检测到三或四种硫醇。由于灵敏度较高（所有硫醇的 LOD 为 0.3 pM），因此也可以定量分析过滤在组织中的与白蛋白结合的低分子量硫醇。
