O'Shea M G, Morell M K
Cooperative Research Centre for Plant Science, Canberra, ACT, Australia.
Electrophoresis. 1996 Apr;17(4):681-6. doi: 10.1002/elps.1150170410.
A novel electrophoretic method for the analysis of oligosaccharides using DNA sequencer technology is illustrated using malto-oligosaccharide distributions obtained following isoamylase digestion of glycogen, wheat starch and potato starch. The debranched starches were derivatized at the reducing and with the charged fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). This highly reproducible method provides baseline resolution of oligomers from chain lengths of 3 to more than 80 glucose units, and exhibits high sensitivity with detection thresholds of one femtomole per resolved band. In addition, the reductive amination procedure attaches a single fluorophore per oligosaccharide, allowing calculation of the results on either a mass or a molar basis. The efficacy of the method is illustrated through the determination of the profile of individual oligosaccharides of chain length with a degree of polymerization (DP) < 80, derived from loading less than 15 ng per analysis of glycogen, wheat and potato starches. While the results obtained were superior in resolution and sensitivity to previously reported observations using a range of techniques, they were nonetheless consistent with the overall differences between these polysaccharides. The resolution, sensitivity, reproducibility and high throughput of the method provides substantial advantages over existing methods for the analysis of linear oligosaccharide chain length distributions.
本文通过对糖原、小麦淀粉和马铃薯淀粉经异淀粉酶消化后得到的麦芽寡糖分布情况进行分析，展示了一种利用DNA测序仪技术分析寡糖的新型电泳方法。将去分支的淀粉在还原端用带电荷的荧光团8-氨基-1,3,6-芘三磺酸（APTS）进行衍生化。这种高度可重复的方法可对链长从3个到80多个葡萄糖单位的寡聚物实现基线分离，并且具有高灵敏度，每个分离条带的检测阈值为1飞摩尔。此外，还原胺化程序使每个寡糖连接一个荧光团，从而能够以质量或摩尔为基础计算结果。通过测定链长小于80且聚合度（DP）的各个寡糖的图谱，展示了该方法的有效性，每次分析糖原、小麦和马铃薯淀粉的上样量小于15 ng。虽然获得的结果在分辨率和灵敏度方面优于先前使用一系列技术所报告的观察结果，但它们与这些多糖之间的总体差异是一致的。该方法的分辨率、灵敏度、可重复性和高通量相较于现有的线性寡糖链长分布分析方法具有显著优势。
