The College of Life Sciences, Northwest University, Xi'an, 710069, China.
Glycobiology and Glycotechnology Research center, College of Food Science and Technology, Northwest University, Xi'an, 710069, China.
Glycoconj J. 2020 Apr;37(2):165-174. doi: 10.1007/s10719-020-09909-z. Epub 2020 Feb 5.
The study of carbohydrates requires large amounts of glycans. N-Glycans can be synthesized but generating large quantities of N-glycans with diverse structures remains difficult. In this study, we aimed to obtain large amounts of glycans using an optimized procedure. Two types of reductive N-glycans were released from chicken egg albumin (ovalbumin) and soy protein using an ammonia catalysis method and labeled with benzenesulfonyl hydrazide (BSH). After preliminary separation by preparative HPLC, N-glycan-BSH components were de-labeled separately and reducing N-glycans were recovered. The de-labeled reducing N-glycans were derived with different labeling reagents and further separated and purified with two/multi-dimensional HPLC for various studies. We selected the bifunctional reagent 2-amino-N-(2-aminoethyl)-benzamide (AEAB) as a labeling reagent combined with C18 column for two-dimensional HPLC separation. A total of 21 and 8 N-glycan-AEAB conjugates were obtained from ovalbumin and soy protein, respectively. A reactive primary alkylamine of N-glycan-AEAB conjugates can be effectively immobilized on microarray surfaces, allowing for subsequent functional studies of glycans.
碳水化合物的研究需要大量的聚糖。N-聚糖可以合成，但生成具有多种结构的大量 N-聚糖仍然很困难。在这项研究中，我们旨在使用优化的程序获得大量的聚糖。使用氨催化方法从鸡卵白蛋白（卵清蛋白）和大豆蛋白中释放出两种类型的还原 N-聚糖，并使用苯磺酰基腙（BSH）标记。通过制备型 HPLC 初步分离后，分别对 N-糖-BSH 组分进行脱标记，回收还原 N-聚糖。脱标记的还原 N-聚糖分别用不同的标记试剂衍生化，并用二维/多维 HPLC 进一步分离和纯化，用于各种研究。我们选择双功能试剂 2-氨基-N-(2-氨基乙基)-苯甲酰胺（AEAB）作为标记试剂，并与 C18 柱结合用于二维 HPLC 分离。从卵清蛋白和大豆蛋白中分别获得了 21 个和 8 个 N-聚糖-AEAB 缀合物。N-聚糖-AEAB 缀合物的反应性伯胺基可有效地固定在微阵列表面上，从而可以对聚糖进行后续的功能研究。
