Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection.

Abstract

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH~=13) has been found to efficiently separate neutral oligosaccharides (triose to undecaose) according to molecular size, sugar composition, and linkage of monosaccharide units. The method was able to resolve 1 -* 3, 1 -> 4, and 1 -* 6 positional isomers of neutral oligosaccharides, which are dermed as having the same number, type, sequence, and anomeric configurations of monosaccharides but differing in the linkage position of a single sugar. From correlating structural features of different oligosaccharides and retention times, we deduced that at least two factors are operative to determine the superior resolution of oligosaccharides by this type of chromatography: (i) the relative acidities ofthe hydroxyl groups and (ii) the accessibility of oxyanions of the oligosaccharides to the functional groups of the stationary phase. Splitting of peaks attributable to mutarotation was not observed. Reducing oligosaccharides were much more retained than their reduced counterparts. Linkage of Fuc(al-3) to GlcNAc of oligosaccharides markedly decreased retention times. Positional isomers of two branched nonosaccharides, which differed by 1 -*6 and 1 -*4 linkages, were widely separated. The separation of 1 -> 3 and 1 -* 4 positional isomers of both tetrasaccharides and glycopeptides containing undecasaccharides demonstrated the significant improvement in resolution of HPAE compared to previous chromatographic methods by either reverse-phase or aminebonded stationary phases. Picomole quantities of underivatized oligosaccharides have been detected by triple-pulse amperometric detection, which produced similar responses for a wide range of structures. Quantification of two triantennary glycopeptides from bovine fetuin by using either detector response or 'H NMR was comparable. The N-glycanase-catalyzed release of two 1 -* 4 and 1 -* 3 positional isomers of an undecasaccharide from a tryptic glycopeptide of bovine fetuin could be observed and quantified by direct injection of the enzyme mixture into the chromatograph.Complex carbohydrates have been implicated in a variety of biological reactions. Cell-cell recognition in development (1) and cancer metastasis (2), intracellular transport oflysosomal enzymes (3), and antibody reactivity to soluble and cellbound carbohydrate determinants (4) are some well-studied examples. Studies designed to elucidate the structural basis of the biological reactivity of naturally derived carbohydrates frequently require the resolution of complex mixtures of oligosaccharides (usually from glycoproteins) or glycolipids. Separation of oligosaccharides that vary only by a single linkage position is often required to define specificities of antibodies (5, 6), lectins (7,8), and glycosyltransferases (9, 10). We report that high-performance anion-exchange chromatography efficiently resolved positional isomers of oligosaccharides and glycopeptides and coupled to electrochemical detection by pulsed amperometry (HPAE-...

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

“…One method relied on electrochemical detection of native, released glycans after separation at high pH: high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). 28,[39][40][41][42] All other methods applied fluorescent labeling. Four methods used electrophoretic separation, which included conventional high-resolution capillary gel electrophoresis with laser-induced fluorescence [CE-LIF(APTS-HR1)], [43][44][45][46][47][48] DNA-sequencer-aided fluorophore-assisted carbohydrate electrophoresis after 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling for high-throughput screening [DSA-FACE(APTS)], [49][50][51][52][53][54] high-resolution capillary gel electrophoresis with rapid labeling with APTS via reductive amination [CE-LIF (APTS-HR2)], and cartridge-based capillary gel electrophoresis with rapid 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) labeling, in development specifically for screening [CCGE(ANTS)].…”

Purification, characterization, and cell wall localization of an α‐fucosidase that inactivates a xyloglucan oligosaccharin

“…in oligosaccharides is known to decrease their HPAE retention time (Hardy and Townsend, 1988), which explains why XG8 (devoid of fucose) has a greater retention time than XG9. Recently, McDougall and Fry (1991a) reported that XG8 does in fact elute later than XG9 on a CarboPak PA-1 anion exchange column.…”

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

“…4. Previously, the components and structures of N-glycans from bovine fetuin have been well characterized by chromatographic separation, HPAEC-PAD analysis, and detailed NMR studies (67)(68)(69)(70). The majority (Ͼ80%) of the N-glycans were triantennary N-glycans with varied degrees of ␣2,6-and/or ␣2,3-sialylations at the terminus.…”

“…5B). The assignment of the peaks was based on a detailed HPAEC-PAD analysis of fetuin N-glycans reported previously (67,69 (Fig. 5C).…”