Identification and Relative Quantification of N-Glycan Structures in Glycoproteins

Glycosylation modification is one of the most common post-translational modification methods in cells, which affects the structure, activity, and localization of proteins through covalent binding of sugar chains, endowing proteins with diverse biological functions and participating in the regulation of various life processes. Depending on the type of sugar chain bound, it can be divided into N-glycosylation and O-glycosylation. N-glycosylation involves the covalent attachment of N-glycans to the asparagine residues of specific amino acid sequences in proteins and is the most well-known form of glycosylation in biopharmaceuticals, especially monoclonal antibodies. The structure of N-glycans influences pharmacokinetics, pharmacodynamics, and immunogenicity, further affecting their efficacy, making the characterization and analysis of N-glycan structures an indispensable part of biopharmaceutical development.

The identification of N-glycoprotein glycans is a complex and challenging task, requiring the analysis of glycan structures including whether specific sites are linked with various glycans, the order of glycosyl residues, the configuration of glycan isomers, the form of glycan cyclization, the linkage between glycans, and branching characteristics. Currently, no single technology can solve all these problems, nor are there conditions for large-scale direct analysis of glycan composition and structure. Common methods for analyzing glycan structures include chromatography, tandem mass spectrometry, and nuclear magnetic resonance.

Compared to structural analysis of glycans, the identification of glycan content is much easier. To determine the average glycan content or glycosylation site occupancy of glycoproteins, MALDI-TOF-MS can be used to directly measure the relative molecular mass of glycoproteins. Glycosidases can be used to cleave glycans and obtain the relative molecular mass of the protein portion. Using the average relative molecular masses of these two parts, the average glycan content of the glycoprotein can be determined. Additionally, traditional SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography can also be used for glycan content identification.

BioPark adopts Thermo Fisher's Q ExactiveHF mass spectrometry platform combined with Nano-LC to provide rapid and efficientNglycanproteomics analysisservice technology package, which can qualitatively and quantitatively identify N-type glycoproteins in mixed systems, including N-glycosylation site, N-glycan structure analysis, and N-glycan content, and also offer customized analysis solutions to meet various detection needs. Free consultation is welcome.

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