The Application of Three-Dimensional HPLC to the Identification of N-Linked Oligosaccharide Structures

Abstract

High-performance liquid chromatography (HPLC) is a useful tool for identifying the structures of N-linked oligosaccharides. And identifying oligosaccharides by examining their elution behaviour on three different columns under three different sets of HPLC conditions, the so-called three-dimensional (3D) mapping technique, is very powerful and convenient. One of the most popular precolumn derivatization methods for oligosaccharides is to label the reducing ends of the oligosaccharides with 2-aminopyridine to provide fluorogenic properties (1,2). N-linked neutral and sialyl pyridylamino (PA)-oligosaccharides are separated by HPLC using three different columns, and their elution positions are expressed in terms of glucose equivalents (3). More than 250 neutral oligosaccharides (4, 5) and over 150 sialyl oligosaccharides (15–17) have been documented on the 2D or 3D map. Many N-linked oligosaccharide structures have been determined using this method, for example, human immunoglobin G (IgG) (6), murine lymphocytes (7), the nicotinic acetylcholine receptor from Torpedo californica (8), recombinant erythropoietin (9), human urinary kallikrein (10), the sphingolipid activator proteins (saposins A, C and D) (11), duck ovomucoid (12), the taste-modifying protein (miraculin) (13) and rice α-amylase (14), human serum (16), human coagulation factor X (17) and many other sources.