Studying the structural and biological function of glycans is essential to understand the mechanism of various biological activities. CD BioGlyco has developed a series of outstanding Enzymatic Cleavage methods by integrating various advanced technologies to provide Glycan Release services.
N-Glycosylation is a common post-translational modification that affects glycoprotein structure and function. N-Glycans are attached to the nitrogen (N) atom in the asparagine (Asn) side chain of the sequon. Glycomic analysis of these intact N-glycans has the potential to lead to therapeutic targets and potentially to treat many genetic defects and their diseases. For example, comparing the glycan profiles of patients and healthy individuals to indicate disease and possible treatment pathways. Studying N-glycans relies on their intact release from glycoproteins, followed by mass spectrometry (MS) techniques analysis.
Fig.1 Workflow for the analysis of N-glycans in glycoproteins. (Regan, et al., 2019)
There are 2 common methods to release N-glycans: chemical release and enzymatic release. Peptide: N-glycosidase F (PNGase F) is the most widely used method for N-glycan release, which releases intact N-glycans to keep the peptide backbone intact. Endoglycosidase H (Endo H) only releases high-mannose N-glycans or hybrid N-glycans. Glycopeptidase A (PNGase A) cleaves all high-mannose, hybrid, and short-complex N-glycans, but fails to release glycans containing sialic acid residues. Hydrazinolysis and β-elimination are the two main chemical release techniques. Both methods are used to release intact glycans from the peptide backbone, but they result in the degradation of the peptide backbone. Therefore, enzymatic methods have become the method of choice for N-glycan release.
The potential applications of N-linked glycosylation in the pharmaceutical field are becoming increasingly apparent. Many therapeutic antibodies on the market are N-linked glycoproteins. In addition, researchers have genetically engineered therapeutic glycoproteins with human-like N-linked glycans. Therefore, the development of efficient glycan release services is necessary.
N-Glycans are essential for physiological and pathological processes. A reliable method must be identified to better separate N-glycans from proteins before analysis. Conventional methods include enzymatic release methods and Chemical Release methods. At CD BioGlyco, we have developed an advanced glycan release platform to provide clients with efficient and accurate N-glycan release services on antibodies, fusion proteins, and other N-glycosylated proteins via PNGase F. We successfully hydrolyze the amide bond between the Asn and N-glycans (including high-mannose, complex, and hybrid N-glycans) to release intact glycans. In addition, we provide clients with fast and sensitive N-glycan quantification and characterization services.
Fig.2 The process of enzymatic release of N-glycan. (CD BioGlyco)
The development of enzymatic cleavage of N-glycan is a complex process. CD BioGlyco has advanced technology platforms and professional research teams in the field of enzymatic release. Our fast and effective strategy will greatly accelerate the project progress of our clients. Please feel free to contact us for more detailed information.
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