Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives.
Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives.
复制标题
DOI：
10.1093/glycob/cwj037
复制
发表时间：
2006
期刊：
影响因子：
4.3
通讯作者：
D. Studelska;Kari Giljum;L. McDowell;Lijuan Zhang
中科院分区：
文献类型：
--
作者：
D. Studelska;Kari Giljum;L. McDowell;Lijuan Zhang
糖胺聚糖（GAG）是由所有动物细胞产生的线性多糖。GAG通过碳水化合物和蛋白质相互作用与数百种蛋白质结合，例如生长因子、细胞因子、趋化因子、细胞外基质组分、蛋白酶抑制剂、蛋白酶和脂蛋白脂肪酶。这些相互作用控制着许多多细胞过程。在生物测定和结合实验中，越来越多地使用从细胞和小组织样品中分离的GAG，这需要灵敏且稳健的定量方法。我们已经开发了这样一种方法，它是基于一个流行的测定氨基酸分析。我们对其进行了改进，以增强GAG定量。它允许定量的葡萄糖胺和半乳糖胺含有糖胺后，其荧光异吲哚衍生物的反相分离。通过邻苯二甲醛和3-巯基丙酸（3 MPA）与GAG酸水解和硼氢化钠还原产生的己糖胺醇单糖的氨基反应产生衍生物。我们的方法的优点包括自动衍生化，一个简单的色谱与清洁分离的氨基葡萄糖醇和氨基半乳糖醇衍生物与污染的氨基酸，良好的灵敏度与0.04 pmol检测，和线性从2.5到1280 pmol。一个主要的优点是，它可以很容易地实施在任何实验室与典型的反相高效液相色谱（HPLC）设备。
Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.