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General microarray technique for immobilization and screening of natural glycans.

General microarray technique for immobilization and screening of natural glycans. We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Analytical Chemistry Pubmed

General microarray technique for immobilization and screening of natural glycans.

Analytical Chemistry , Volume 79 (21): -8093 – Dec 21, 2007

General microarray technique for immobilization and screening of natural glycans.


Abstract

We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins.

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ISSN
0003-2700
DOI
10.1021/ac071187g
pmid
17922555

Abstract

We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins.

Journal

Analytical ChemistryPubmed

Published: Dec 21, 2007

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