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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-...

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Glycopeptides were isolated from bovine fetuin after digestion with Pronase, aminopeptidase M, and carboxypeptidase Y. The glycopeptides were derivatized with tert-butyloxycarbonyltyrosine and separated on the basis of peptide by using reverse-phase high-performance liquid chromatography. Using 400-MHz 1H NMR, the asialotriantennary oligosaccharides at each of the three N-linked glycosylation sites were found to be combinations of the following two structures in which the third branch is either Gal beta(1,4)GlcNAc or Gal beta(1,3)GlcNAc: (formula; see text) The asialotriantennary glycopeptides containing all beta(1,4)-lactosamine as the branches were designated Gal beta(1,4)GlcNAc-TRI while triantennary glycopeptides containing beta(1,3)-lactosamine as branch III were termed Gal beta(1,3)GlcNAc-TRI. The Gal beta(1,3)GlcNAc unit was localized predominantly to the branch III arm on the basis of a downfield shift (-0.027 ppm) in the H-1 and upfield shift (0.01 ppm) in the NAc methyl signals from the branch III GlcNAc resulting from Gal beta(1,3) instead of Gal beta(1,4) substitution. Revised assignments are proposed for the H-1's of Gal residues 6 (delta 4.464) and 8 (delta 4.471) [Vliegenthart, J. F. G., Dorland, L., & van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209-373] in a Gal beta(1,4)GlcNAc-TRI. The proportion of Gal beta(1,3)GlcNAc-TRI glycopeptides from the Asn-Asp, Asn-Gly, and Asn-Cys sites was found to be 40%, 60%, and 20%, respectively. Analysis of the binding of these glycopeptides, containing from 20% to 60% Gal beta(1,3)GlcNAc as branch III, to rabbit hepatocytes revealed that the greater the proportion of Gal beta(1,3)GlcNAc, the lower the affinity of the mixture. The Kd for Gal beta(1,4)GlcNAc-TRI was found to be between 3.6 and 5.4 nM (P = 0.10) with a mean of 4.4 nM from binding data analyzed by using the LIGAND program [Munson, P. J., & Rodbard, D. (1980) Anal. Biochem. 107, 220-239] and computer simulations of the binding of two ligands as a mixture to one receptor site. The Kd of Gal beta(1,3)GlcNAc-TRI oligosaccharide, prepared by hydrazinolysis, was found to be 305 nM from inhibition studies.

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