A Rapid High-Resolution High-Performance Liquid Chromatographic Method for Separating Glycan Mixtures and Analyzing Oligosaccharide Profiles

Guile, Geoffrey R.; Rudd, Pauline M.; Wing, David R.; Prime, Sally B.; Dwek, Raymond A.

doi:10.1006/abio.1996.0351

Cited by 506 publications

(462 citation statements)

References 8 publications

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“…The enzymatic modifications include de-sialylation, de-galactosylation, de-N-acetylglucosaminosylation and de-mannosylation at various levels using exoglycosidases, synthesis of Gal(α1-3)Gal epitopes using α-1,3-galactosyltransferase, and removal of N-linked glycans using endo-β-Nacetylglucosaminidase Endo-H and amidase PNGase F (peptide-N 4 -(N-acetyl-β-glucosaminyl) asparagine amidase F). The reference proteins and their variants were fully characterized using chromatographic [13] and mass spectrometric [14] methods, in cases where information from the literature was not available. We then created thousands of fingerprints of several hundreds of glycoform mixtures, derived from the glycoproteins.…”

Section: Resultsmentioning

confidence: 99%

A lectin array-based methodology for the analysis of protein glycosylation

Rosenfeld¹,

Bangio²,

Gerwig³

et al. 2007

Journal of Biochemical and Biophysical Methods

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Glycosylation is the most versatile and one of the most abundant protein modifications. It has a structural role as well as diverse functional roles in many specific biological functions, including cancer development, viral and bacterial infections, and autoimmunity. The diverse roles of glycosylation in biological processes are rapidly growing areas of research, however, Glycobiology research is limited by the lack of a technology for rapid analysis of glycan composition of glycoproteins. Currently used methods for glycoanalysis are complex, typically requiring high levels of expertise and days to provide answers, and are not readily available to all researcher.We have developed a lectin array-based method, Qproteome™ GlycoArray kits, for rapid analysis of glycosylation profiles of glycoproteins. Glycoanalysis is performed on intact glycoproteins, requiring only 4-6 h for most analysis types. The method, demonstrated in this manuscript by several examples, is based on binding of an intact glycoprotein to the arrayed lectins, resulting in a characteristic fingerprint that is highly sensitive to changes in the protein's glycan composition. The large number of lectins, each with its specific recognition pattern, ensures high sensitivity to changes in the glycosylation pattern. A set of proprietary algorithms automatically interpret the fingerprint signals to provide a comprehensive glycan profile output.

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Section: Resultsmentioning

confidence: 99%

A lectin array-based methodology for the analysis of protein glycosylation

Rosenfeld¹,

Bangio²,

Gerwig³

et al. 2007

Journal of Biochemical and Biophysical Methods

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“…Glycans were removed by hydrazinolysis using a Glycoprep 1000 machine (Oxford Glycosciences, Abingdon, Oxfordshire, U.K.) and fluorescently labeled at their reducing end with 2-aminobenzamide using the Signal Labeling Kit (Oxford Glycosciences). Glycans were subsequently analyzed by weak anion exchange (WAX) chromatography (12) and normal phase HPLC (13). Glycan structures were identified using sequential exoglycosidase digestion as described (13).…”

Section: Methodsmentioning

confidence: 99%

“…Glycans were subsequently analyzed by weak anion exchange (WAX) chromatography (12) and normal phase HPLC (13). Glycan structures were identified using sequential exoglycosidase digestion as described (13).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: Evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion

Mehta

Block

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…Normal Phase Chromatographic Separations of Neutral and Acidic Oligosaccharides-Separations were performed on a GlycoSep-N HPLC column (Oxford GlycoSystems, Ltd.) (4.6 ϫ 250 mm) as described previously (32).…”

Section: Methodsmentioning

confidence: 99%

“…The assignment of each of the main peaks in the CD59 glycan pool was checked by following its predicted elution position through each of the enzyme digests. Predicted elution positions were based on predetermined incremental values for monosaccharide additions to standard glycan cores (32) and the known specificity of the enzymes (Fig. 1b).…”

Section: Unit Values and Structures Assigned To The N-linked Glycans mentioning

confidence: 99%