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N-Glycan Analysis

N-Glycan Analysis

As one of the most important post-translational modifications of proteins in eukaryotic cells, protein glycosylation is involved in a wide range of biological and physiological processes, including recognition & regulatory functions, cellular communication, gene expression, cellular immunity, growth and development.

Glycan functions are usually determined by the structures of the oligosaccharides, which are covalently attached to proteins primarily at 2 structural motifs: the amide group of an asparagine (N-glycans) or the hydroxyl group on serine or threonine (O-glycans). Because of the diversity of the oligosaccharides, even glycosylation at a single site can generate considerable heterogeneity of the mass and charge of glycoproteins. Although different approaches for N-glycans analysis have been described, usually these methods are based on enzymatic release of N-glycans from the protein by PNGase F, and derivation of released glycans, due to the lack of intrinsic chromophores, with a fluorescent labelling before analysis.

As the largest group of therapeutic proteins, the efficacy of the mAb is highly dependent on the correct glycosylation pattern. In Creative Proteomics, our tech support can help you to characterize the glycosylation of mAb and other glycoprotein therapeutics, using CE-based N-Glycan analysis, which is considered as the golden standard major biotech companies apply for quality control. After separation of major glycan peaks and identification with glycan standards, the relative abundance of the major N-glycan types found on antibodies can be determined.

The workflow for N-glycan analysis is as following:

Deglycosylation to release N-linked glycans by enzymatic cleavage using PNGase F;
Fluorescence labeling of the released glycans in presence of deglycosylated proteins;
Profiling of the released major glycans by capillary electrophoresis/LC-MS

Staffed with experienced bioanalysts and technicians, Creative Proteomics can provide rapid and professional analytical support to your project.


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