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Glycan Analysis

Glycosylation is an enzymatically driven post-translational process by which saccharides are linked to proteins and lipids through glycosydic bond. Glycosylation prevents protein degradation, helps in protein folding and increases the half-life. Understanding the nature and composition of glycans is vital because it influences not only the structural integrity of the protein, but also plays a critical role in biological activity of the proteins, like receptor binding and in-vivo clearance.

In proteins, glycans are classified as N-linked or O-linked based on their attachment to the amide nitrogen of Asparagine (Asn) or to the hydroxyl group of Threonine and/or Serine. The nature and composition of glycans entirely depend on manufacturing conditions.

We offer the following techniques for the Glycan profile analysis of glycoproteins Capillary electrophoresis combined with Laser Induced Fluorescence (LIF) detection provides a very powerful and accurate method for the antennary structure determination of the glycoproteins. Being a capillary separation, the sample requirements are very low at picomole levels. This is very important for identifying the glycoprofile of novel proteomics where the sample availability could be very low.

We offer very well established services for determination antennary structure for known and unknown proteins.

NPLC-FLD

N-glycans are enzymatically removed by PNGase F and recovered using EB10 purification column. The purified N-glycans are vacuum dried and labeled with 2-AB (2-amino benzamide). The separation of glycans is achieved by chromatography using Amide based column (Normal Phase). The separation in amide column is based on hydrogen bonding capacity of a glycan. Larger the glycan structure, higher its H-bonding capacity and longer it stays in the column. The identification of N-glycan structure (Antennary) can be achieved by mass spectrometry coupled with enzymatic analysis.

Carbohydrate Analysis

At our facility, carbohydrate analysis is performed using both the traditional HPAEC-PAD and Fluorescence based methods.

HPAEC-PAD method (Dionex ICS-3000)

For analysis of monosacharides such as glucose, fucose, N acetyl galactoseamine, N acetyl glucosamine; glycans are extracted from the protein and subjected to acid hydrolysis. The resulting monosaccharides are separated using High Performance Anion Exchange chromatography and detected by Pulsed Amperometric detector. The separated monosaccharides are identified and quantified against a standard curve for each monosaccharide.

A relatively mild acid hydrolysis method is used for sialic acids which are again quantified against a standard calibration curve of N acetylneuraminic acid and N glycolylneuraminc acid (most commonly observed in glycoprotein).

Fluorescence based method

In this method, which is relatively more sensitive; the released monosaccharides and sialic acids (different hydrolysis conditions) are derivatized using a flourophore such as 2 Amino benzoic acid/1, 4- Diaminobutane/2-aminobenzamide and detected using a fluorescence detector. Quantitation of individual monosacharides and sialic acids is done against a respective standard calibration curve.