Glycosylation Site Profiling

Glycosylation site profiling mainly contributes to the illustration of glycan microheterogeneity, which is important for the understanding of glycoprotein biosynthesis and function. As a forward-looking company as well as a market leader in the field of therapeutic antibody development and analysis, Creative Biolabs has successfully accomplished numerous challenging projects in antibody glycan analysis and glycosylation site profiling. Now, we can provide an integrated platform for one-stop glycosylation analysis of your therapeutic antibody candidates. As your best partner, Creative Biolabs is very glad to revolutionize your particular project and promote your success based on the values of quality, timing, and price.

Glycosylation is Necessary for Antibody Property

Therapeutic monoclonal antibodies (mAbs) exhibit heterogeneity in structure and function because of the complex posttranslational modifications, including glycosylation. It has been documented that glycosylation, especially Fc glycosylation, plays a critical role for the determination of antibody physicochemical properties (solubility, viscosity, protein folding, antigen binding, secretion, immunogenicity, etc.) and biological properties [antibody-dependent-cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)]. Therefore, antibody glycan analysis should be determined to produce and develop human mAb drugs with proper function, efficacy, safety, stability, and consistency.

Fig.1 Schematic workflow exemplifying the glycan analysis of a complex glycoprotein.¹

Glycosylation Site Profiling Constitute Glycosylation Analysis

The profile of glycosylation sites is an important component of glycosylation analysis, which can supplement already obtained data on protein structure. The site information provides a basis for future detailed characterization regarding the in vivo relevance of the respective site. Furthermore, glycosylation sites may indicate the orientation of transmembrane proteins because glycosylations are commonly encountered at the extracellular domains of proteins. Therefore, glycosylation site analysis is routinely performed on biopharmaceutical products (such as mAbs and glycoproteins) throughout their development and production processes.

Fig.2 Workflow of the glycosylation sites mapping. (Ying, 2017)

Technologies for Glycosylation Site Profiling

Accurate characterization of glycosylation sites is needed for understanding the functions of glycans in mAbs. To determine the site of glycosylation and the glycans on the glycosylation site, first of all, glycoproteins must be cleaved into small glycopeptides. The enzymes trypsin (a highly specific protease) and (non-specific) are widely used in this process. Then, the glycans and peptide sequence on the glycosylation site can be determined by mass spectrometry (MS), which has been the most powerful tool for glycosylation site identification, occupancy measurement, and site-specific glycan structure characterization. At Creative Biolabs, we can provide a series of methods to perform glycosylation site profiling, including but not limited to the followings.

• RP-HPLC-MS
  Reversed-phase high-performance liquid chromatography coupled with on-line mass spectrometry (RP-HPLC-MS) can analyze the fractionated peptide and glycopeptides fragments after digestion of a glycoprotein with specific proteases after reduction and alkylation. This method is semi-quantitative because different glycopeptides will ionize differently in a mass spectrometer.
• LC-ESI-MS
  Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) is a hyphenated mass spectrometry technique that combines the resolving power of HPLC separation with high mass accuracy of a mass spectrometer. It provides unparalleled sensitivity and selectivity for detection and quantification of site-specific glycan structure.
• HILIC-SPE-MS
  Solid-phase extraction using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-SPE-MS) has become increasingly popular in the glycosylation site mapping because HILIC-SPE-MS is more selective for glycosylated species and is not biased toward particular glycan types. Furthermore, using that technique requires no chemical alteration of glycans.
• ESI/ETD-CID-MS/MS
  Electrospray-ionization/Electron transfer dissociation, collision-induced dissociation, tandem mass spectrometry (ESI/ETD-CID-MS/MS) allows for fragmentation of glycan species to be observed predominantly at the glycosidic linkages while the peptide backbone remains intact. CID provides information about the glycan composition.

Why Choose Us?

• High throughput with sensitivity and specificity
• High batch-to-batch consistency
• One-stop service with fully customized design
• Competitive prices with best after-sale service

Characterization of glycosylation sites is important for the understanding of glycosylated antibody biosynthesis and function. Backed by extensive expertise and state-of-the-art technologies, Creative Biolabs has developed, optimized, and validated a panel of technology platforms to offer high-quality glycosylation site analysis services to our global clients. Additionally, we can offer fully customized study designs to meet your specific demands. If you are interested in our services, please contact us or directly sent us an inquiry.

References

1. Mastrangeli, Renato, Abhijeet Satwekar, and Horst Bierau. "Innovative Metrics for Reporting and Comparing the Glycan Structural Profile in Biotherapeutics." Molecules 28.8 (2023): 3304.
   Distributed under Open Access License CC BY 4.0, without modification.
2. Ying, Jiezheng, et al. "Mapping the N-linked glycosites of rice (Oryza sativa L.) germinating embryos." PLoS One 12.3 (2017): e0173853.
   Distributed under Open Access License CC BY 4.0, without modification.

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