Structure analysis of glycoproteins by two dimensional HPLC mapping with high sensitivity

糖蛋白糖链的结构分析通过以下策略通过二维HPLC图谱以高灵敏度进行。糖蛋白的糖链通过肼解而被释放，游离的氨基被重新乙酰化。用NaBH_3CN将糖链的还原端与2-氨基吡啶偶联。由此获得的糖链的吡啶基氨基（PA-）衍生物通过以下色谱法分离：1）DEAE-HPLC在DEAE-HPLC的条件下，PA-糖链根据唾液酸残基的数目分离。2）尺寸分级HPLC在尺寸分级HPLC的条件下，PA-糖链根据其分子大小分离。3）反相HPLC在反相HPLC的条件下，PA-糖链根据其PA-糖链的结构如异头构型、键的类型和糖残基而分离，并且不受其分子大小的很大影响。PA-糖链被很好地分离，并且通过比较PA-糖链与标准PA-糖链的洗脱时间来评估它们的结构。4）结合外切糖苷酶的酶切研究和洗脱时间与PA糖链结构的规律，进一步证实了该方法的结构。

Structure analysis of sugar chains of glycoproteins is performed with high sensitivity by two dimensional HPLC mapping by the following strategy. Sugar chains of glycoproteins are liberted by hydrazinolysis and the freee amino groups are re-acetylated. The reducing ends of the sugar chains are coupled with 2-aminopyridine by means of NaBH_3CN. The pyridylamino (PA-) derivatives of sugar chains thus obtained are separated by the following chromatographies;1) DEAE-HPLCUnder the conditions of DEAE-HPLC, PA-sugar chains are separated according to the number of sialic acid residues.2) Size-fractionation HPLCUnder the conditions of size-fractionation HPLC, PA-sugar chains are separated according to their molecular sizes.3) Reversed-phase HPLCUnder the conditions of reversed-phase HPLC, PA-sugar chains are separated according to their structures of PA-sugar chains such as anomeric configurations, type of linkages, and sugar residues and not influenced largely by their molecular sizes.By using the HPLC as described under 2) and 3), PA-sugar chains are well separated and their structures are assessed by comparing the elution times of PA-sugar chains with those of standard PA-sugar chains.4) The structures are further confirmed by combination of exoglycosidase digestion studies and the rule between the elution times and structures of PA-sugar chains.This method was applied to structure analysis of sugar chains from natural interferon- .