ANALYSES OF N-LINKED OLIGOSACCHARIDES USING A TWO-DIMENSIONAL MAPPING TECHNIQUE
ANALYSES OF N-LINKED OLIGOSACCHARIDES USING A TWO-DIMENSIONAL MAPPING TECHNIQUE
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DOI：
10.1016/0003-2697(88)90126-1
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发表时间：
1988-05-15
影响因子：
2.9
通讯作者：
TAKAHASHI, N
中科院分区：
文献类型：
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作者：
TOMIYA, N;AWAYA, J;TAKAHASHI, N
我们提出了一个简单的，可重复的，灵敏的分析N-连接寡糖的结构的二维糖图方法。未知寡糖的结构可以从其在图谱上的位置来表征。利用113种标准寡糖制备了糖图谱数据库，其中58种寡糖的结构已被1H NMR谱所证实。本方法包括六个步骤，（i）通过N-寡糖糖肽酶（杏仁）消化从糖肽制备寡糖，（ii）通过使用氰基硼氢化钠，用荧光试剂2-氨基吡啶衍生寡糖的还原末端，（iii）通过ODS-硅胶柱的高效液相色谱分离寡糖衍生物，（iv）在酰胺-硅胶柱上分析每种分离的寡糖的大小，（v）在标准寡糖获得的二维糖图谱上绘制样品的洗脱位置，和（vi）通过连续外切糖苷酶消化和步骤（iii-v）的组合分析寡糖的结构。将该方法应用于鸡卵清蛋白中寡糖的结构鉴定。结果发现卵清蛋白中存在两种未见报道的低聚糖。
We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.