Ultra-sensitive Analysis of Carbohydrates

(Update Issue: April.23, 2007)
 Highly specific and sensitive detection using high-resolution separation mode is required for the analysis of micro amounts of oligosaccharides derived from glycoconjugates such as glycoproteins or glycosaminoglycans. High performance liquid chromatography using a latex-type anion exchange column with pulse amperometric detection on a gold electrode is of primary choice for the analysis of oligosaccharides derived especially from glycoproteins having d.p. 10 to 20 (1). Strongly alkaline solution is used as eluent in this system, and some of the hydroxyl groups of saccharides are dissociated to hydroxy anions. Therefore, oligosaccharides are separated in an anion exchange column based on the number of acidic residues as well as the type of component monosaccharides and their linkage positions. Alkaline eluent also generates reactive oxidants on a gold electrode, catalytically oxidizing hydroxyl groups of saccharides, and the detection is achieved by monitoring the change in electric current due to the oxidation of saccharides. This method does not require preliminary derivatization or any other chemical treatment of saccharide samples. The molar sensitivity is enhanced by increasing the molecular weight because the efficiency of oxidation depends on the number of hydroxyl groups of saccharide samples. However, alkaline mobile phase may cause saccharide degradation such as peeling or epimerization to some extent.

There are many reports concerning fluorescent labeling for the analysis of saccharides (Fig. 1). Many reagents are categorized in a group using a common reaction scheme called reductive amination in which a saccharide is converted to glycamine in the presence of a reducing agent such as sodium cyanoborohydride (NaBH3CN) (Fig. 2). Labeling using 2-aminopyridine (PA) (2), originally reported by Hase et al., has developed to 2-dimensional or 3-dimensional mapping by Takahashi et al. (3, 4). The structures of PA-labeled oligosaccharides from glycoproteins were easily confirmed by plotting the elution data collected on a reversed-phase ODS column, a normal-phase amide column and an ion-exchange DEAE column, respectively. Some acid-labile residues such as sialic acids are released during derivatization. The labeling reagent should be removed prior to the analysis.

ABA:2-Aminobenzoic acid
ABEE:Ethyl p-aminobenzoate
ANTS:8-Aminonaphthalene-1,3,6-trisulfonic acid
ANDS:7-Aminonaphthalene-1,3-disulfonic acid
APTS:8-Aminopyrene-1,3,6-trisulfonic acid
Fig. 1. List of reagents used for labeling of saccharides
Fig. 2. Fluorescent labeling of saccharides by reductive amination
Diffusion of sample components in the LC system hampers ultramicro-sensitivity. Capillary electrophoresis (CE) using a very narrow capillary (50 µm i.d.) has come to the front. Aminonaphthalenetrisulfonate (ANTS) and aminopyrenetrisulfonate (APTS) have strong fluorescence, and also strong anionic characteristics, enhancing the resolution of their carbohydrate derivatives in CE. Guttman et al. reported the excellent separation of APTS derivatives derived from various glycoproteins (5, 6). Sensitive detection in zeptomole (10-21 mol) level in CE using rhodamine derivatives has made it possible to analyze the metabolism of carbohydrates in a single cell (7).
Shigeo Suzuki (Faculty of Pharmaceutical Sciences, Kinki University)
References (1) Townsend, RR, Hardy, MR, Lee, YC : Separation of oligosaccharides using high-performance anion exchange chromatography with pulsed amperometric detection. Methods Enzymol. 179, 65-76, 1989
(2) Hase, S, Ikenaka, T, Matsushima, Y : Structure analysis of oligosaccharides by tagging of the reducing end sugars with a fluorescent compounds. Biochem. Biophys. Res. Comm. 85, 257-263, 1978
(3) Tomiya, N, Kurono, M, Ishihara, H, Tejima, S, Endo, S, Arata, Y, Takahashi, N : Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography. Anal. Biochem. 163, 489-499, 1987
(4) Nakagawa, H, Kawamura, Y, Kato, K, Shimada, I, Arata, Y, Takahashi, N : Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography. Anal. Biochem., 226, 130-138, 1995
(5) Guttman, A : Multistructure sequencing of N-linked fetuin glycans by capillary gel electrophoresis and enzyme matrix digestion. Electrophoresis, 18, 1136-1141, 1997
(6) Guttman, A, Herrick, S : Effect of the quantity and linkage position of mannose(alpha1,2) residues in capillary gel electrophoresis of high-mannose-type oligosaccharides. Anal. Biochem. 235, 236-239 1996
(7) Le, XC, Tan, W, Scaman, CH, Szpacenko, A, Arriaga, E, Zhang, Y, Dovichi, NJ, Hindsgaul, O, Palcic, MM : Single cell studies of enzymic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast. Glycobiology, 9, 219-225, 1999
Dec.15, 1999

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