Analysis of Oligosaccharide Structures from the Reducing End Terminal by Combining Partial Acid Hydrolysis and a Two-Dimensional Sugar Map

结构分析是阐明糖蛋白糖链作用的必要步骤。然而，从天然来源分离的具有生物活性的糖蛋白通常只能以有限的量获得。为了克服这一缺点，我们开发了一种荧光标记方法，我们一直使用与外切糖苷酶消化相结合。尽管这种方法很有用，但我们经常遇到在获得酶或在孵育条件下的困难，即使我们有酶，由于底物特异性，它也不总是能够释放糖残基。为了克服这个问题，开发了一种新的灵敏和方便的寡糖结构分析方法，其中部分酸水解与吡啶氨基（PA-）寡糖的二维糖图谱相结合。对APA-寡糖进行了部分水解，并对酸水解条件进行了优化，以从不同类型的PA-寡糖中获得高产率的各种PA-寡糖片段。然后通过大小分级HPLC每1个葡萄糖单元分级酸水解产物，并且通过反相HPLC分析每个级分。在用标准PA-糖链制备的二维图谱上鉴定每个PA-寡糖片段的结构，之后基于所获得的PA-寡糖片段的结构从还原末端重构原始PA-寡糖。该方法已成功地应用于解析N-连接糖链的结构。

Structure analysis is a necessary step in elucidating the roles of glycoprotein sugar chains. Glycoproteins with biological activity isolated from natural sources, however, are often obtainable in only limited amounts. To circumvent this drawback, we developed a fluorescence labeling method, which we have been using in combination with exoglycosidase digestion. Although the method is useful, we frequently encounter difficulty in obtaining enzymes or in the incubation conditions, and even when we do have an enzyme, it is not always able to liberate sugar residues due to substrate specificity.To overcome this, a new sensitive and convenient method for the structural analysis of oligosaccharides was developed, in which partial acid hydrolysis was combined with two-dimensional sugar mapping of pyridylamino (PA-) oligosaccharides. APA-oligosaccharide was partially hydrolyzed, the acid hydrolysis conditions being optimized so as to obtain various PA-olgosaccharide fragments with high yields from different types of PA-oligosaccharides. The acid hydrolyzates were then fractionated every 1 glucose unit by size-fractionation HPLC, and each fraction was analyzed by reversed-phase HPLC. The structure of each PA-oligosaccharide fragment was identified on a two-dimensional map prepared with standard PA-sugar chains, after which the original PA-oligosaccharide was reconstructed from the reducing end terminal based on the obtained structures of the PA-oligosaccharide fragments. The method was successfully applied to resolving the structure of an N-linked sugar chain.