Takahashi N, Wada Y, Awaya J, Kurono M, Tomiya N
GlycoLab, Nakano Central Research Institute, Nakano Vinegar Co., Ltd., Handa City, Japan.
Anal Biochem. 1993 Jan;208(1):96-109. doi: 10.1006/abio.1993.1013.
We have previously published a two-dimensional (2-D) mapping technique for N-linked oligosaccharides using pyridylaminated derivatives (PA-oligosaccharides) (N. Tomiya et al. Anal. Biochem. 171, 73-90, 1988). We now report an extension of this method to GalNAc-containing N-linked oligosaccharides. The new 2-D map was prepared from the elution data of 40 different GalNAc-containing oligosaccharides, 16 of which were obtained directly from human urinary kallidinogenase by digestion with glycopeptidase A. The other 24 oligosaccharides were derived by subsequent digestion of the 16 original oligosaccharides with beta-galactosidase or alpha-fucosidase. Each of the 40 oligosaccharide derivatives was separated by high-performance liquid chromatography using ODS-silica and amide-silica columns. The 2-D map constructed by plotting elution position of each oligosaccharide (expressed in terms of glucose units) can be useful as such in delineating the structure of an unknown oligosaccharide by direct placement of its elution positions in the 2-D map. Multiple regression analysis of the data as performed previously yielded parameters related to the contribution of each component monosaccharide unit to the elution profile. The best results were obtained when the GalNAc-containing PA-oligosaccharides were classified into an F-series (those containing Fuc alpha 6GlcNAc-PA) and a Z-series (all others), based on our previous classification method. These calculated values are useful in predicting oligosaccharide structure from known elution values as well as to predict elution volumn from a known structure. The structure of a minor GalNAc-containing oligosaccharide in human urinary kallidinogenase was elucidated using these newly calculated values.
我们之前发表了一种使用吡啶氨基化衍生物（PA-寡糖）对N-连接寡糖进行二维（2-D）图谱分析的技术（N. Tomiya等人，《分析生物化学》171，73 - 90，1988）。我们现在报告将该方法扩展到含N-乙酰半乳糖胺（GalNAc）的N-连接寡糖。新的二维图谱是根据40种不同含GalNAc寡糖的洗脱数据绘制而成，其中16种是通过用糖肽酶A消化人尿激肽释放酶原直接获得的。另外24种寡糖是通过随后用β-半乳糖苷酶或α-岩藻糖苷酶消化这16种原始寡糖得到的。40种寡糖衍生物中的每一种都使用ODS-硅胶柱和酰胺硅胶柱通过高效液相色谱进行分离。通过绘制每种寡糖的洗脱位置（以葡萄糖单位表示）构建的二维图谱，可通过将未知寡糖的洗脱位置直接放置在二维图谱中来描绘其结构。如之前所做的那样对数据进行多元回归分析，得到了与每个单糖组成单位对洗脱图谱贡献相关的参数。当根据我们之前的分类方法将含GalNAc的PA-寡糖分为F系列（那些含有Fucα6GlcNAc-PA的）和Z系列（其他所有的）时，获得了最佳结果。这些计算值可用于根据已知洗脱值预测寡糖结构，以及根据已知结构预测洗脱体积。利用这些新计算的值阐明了人尿激肽释放酶原中一种次要含GalNAc寡糖的结构。
