Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Anal Biochem. 2012 Aug 1;427(1):91-8. doi: 10.1016/j.ab.2012.05.004. Epub 2012 May 15.
A quantitative and highly sensitive method for the analysis of glycosaminoglycan (GAG)-derived disaccharides that relies on capillary electrophoresis (CE) with laser-induced fluorescence detection is presented. This method enables complete separation of 17 GAG-derived disaccharides in a single run. Unsaturated disaccharides were derivatized with 2-aminoacridone to improve sensitivity. The limit of detection was at the attomole level and approximately 100-fold more sensitive than traditional CE-ultraviolet detection. A CE separation timetable was developed to achieve complete resolution and shorten analysis time. The relative standard deviations of migration time and peak areas at both low and high concentrations of unsaturated disaccharides are all less than 2.7 and 3.2%, respectively, demonstrating that this is a reproducible method. This analysis was successfully applied to cultured Chinese hamster ovary cell samples for determination of GAG disaccharides. The current method simplifies GAG extraction steps and reduces inaccuracy in calculating ratios of heparin/heparan sulfate to chondroitin sulfate/dermatan sulfate resulting from the separate analyses of a single sample.
本文介绍了一种基于毛细管电泳（CE）激光诱导荧光检测的定量、高灵敏度的糖胺聚糖（GAG）衍生二糖分析方法。该方法可在单次运行中完全分离 17 种 GAG 衍生二糖。非饱和二糖用 2-氨基吖啶酮衍生化以提高灵敏度。检测限达到了飞摩尔水平，比传统的 CE-紫外检测灵敏约 100 倍。开发了 CE 分离时间表以实现完全分辨率并缩短分析时间。在低浓度和高浓度非饱和二糖时，迁移时间和峰面积的相对标准偏差均小于 2.7%和 3.2%，表明该方法具有良好的重现性。该分析成功应用于培养的中国仓鼠卵巢细胞样品中 GAG 二糖的测定。与单独分析单个样品导致肝素/硫酸乙酰肝素与软骨素硫酸盐/硫酸皮肤素比值计算不准确相比，该方法简化了 GAG 提取步骤，减少了误差。
