Kotani N, Takasaki S
Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
Anal Biochem. 1998 Nov 1;264(1):66-73. doi: 10.1006/abio.1998.2820.
To establish high-pH anion-exchange chromatography with fluorometric detection (HPAEC-FD) for the sensitive oligosaccharide analysis, the system for HPAEC with pulsed amperometric detection (PAD) was modified by placing the anion micromembrane suppressor between PAD and fluoromonitor to neutralize the alkaline eluate and connecting the flow path. N-Linked oligosaccharides were released from various glycoproteins by hydrazinolysis followed by labeling with 2-aminobenzamide, and their asialooligosaccharides were analyzed by HPAEC-FD using an isocratic elution with 0.3 M NaOH solution. The results indicated that the new system using FD offers fine separation of oligosaccharides at the subpicomole level. Coinjection of reduced dextran oligomers as internal standards which are detected by PAD permits us to express elution positions of the fluorescent oligosaccharides as glucose units, resulting in a much more reliable analysis. The system was also effectively used for structural analysis of oligosaccharides by sequential glycosidase digestion. Thus, HPAEC-FD promises to be an alternative tool for the sensitive analysis of oligosaccharides.
为建立用于灵敏寡糖分析的带荧光检测的高pH值阴离子交换色谱法（HPAEC-FD），对带脉冲安培检测（PAD）的HPAEC系统进行了改进，方法是在PAD和荧光监测器之间放置阴离子微膜抑制器以中和碱性洗脱液并连接流路。通过肼解从各种糖蛋白中释放出N-连接寡糖，然后用2-氨基苯甲酰胺进行标记，并用0.3 M NaOH溶液等度洗脱，通过HPAEC-FD分析其去唾液酸寡糖。结果表明，使用FD的新系统能在亚皮摩尔水平对寡糖进行良好分离。共注入经PAD检测的还原葡聚糖低聚物作为内标，使我们能够将荧光寡糖的洗脱位置表示为葡萄糖单位，从而实现更可靠的分析。该系统还通过顺序糖苷酶消化有效地用于寡糖的结构分析。因此，HPAEC-FD有望成为寡糖灵敏分析的替代工具。
