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

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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- .