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

糖蛋白的糖链结构分析采用二维高效液相色谱图谱，具有高灵敏度。糖蛋白的糖链被肼解释放，游离氨基被重新乙酰化。糖链的还原端通过NaBH_3CN与2-氨基吡啶偶联。由此得到的糖链吡啶氨基（PA-）衍生物通过以下色谱分离；1) DEAE-HPLC在DEAE-HPLC条件下，根据唾液酸残基的数量分离pa -糖链。2)粒径分馏高效液相色谱在粒径分馏高效液相色谱条件下，pa -糖链根据其分子大小进行分离。3)反相高效液相色谱法：在反相高效液相色谱条件下，pa -糖链的分离是根据pa -糖链的结构（如异位构型、键类型和糖残基）进行的，不受其分子大小的影响。通过2)和3)所述的高效液相色谱法，pa -糖链被很好地分离，并通过比较pa -糖链与标准pa -糖链的洗脱时间来评估其结构。4)结合外糖苷酶酶切研究和pa -糖链的洗脱时间与结构之间的规律进一步证实了结构。该方法应用于天然干扰素-糖链的结构分析。

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- .