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Glycosylation Analysis

We provide the following services in analysing protein glycosylation:

  • Released N-glycan analysis 

  • N-glycan linkage analysis 

  • site-specific N-glycan analysis

  • site specific O-glycan analysis.


More details of above glycosylation analysis can be found in the following sections.


1. Released N-Glycan Analysis

Gemini Bio offers an efficient service for released N-glycan analysis with HILIC separation and dual detections of N-glycan species. The fluorescence detection of 2-AB labeled glycan species gives a sensitive and quantitative measurement of the ratio of each glycan species;  MS and MS/MS of glycan species provides accurate and detailed information about the glycan structure and a highly confident glycan structure determination.


In released glycan analysis, a glycoprotein is treated with PNGase F to cleave N-glycans off the protein. Optionally, if N-glycans contain negatively charged sugars such as sialic acid at a terminal, we need to use an enzyme to remove them to enhance MS signal intensity. The released N-glycan is derivatized by a chemical reaction with 2-aminobenzamide (2-AB). The labeled glycan is isolated and separated by HPLC with an analytical HILIC column,  which separates labeled N-glycans based on their glucose units. The eluate from the HPLC column is analyzed by a fluorescence detector and mass spectrometry. The chromatogram from HILIC separation and fluorescence detection provide a quantitative, highly reproducible measurement about the type, ratio and glucose unit of N-glycan species. The MS and MS/MS data provides a more accurate determination of each glycan species.

The released N-Glycan analysis is routinely used for the characterization of therapeutic antibodies and proteins.  We adhere to ICH Q6B guideline and we will meet any specific requirements of our clients.

2. The N-Glycan Linkage Analysis

Due to the high complexity of glycan structures of glycoprotein, the N-glycan linkage analysis is sometimes needed to confirm the configuration and position of glycosidic linkages.

In our N-Glycan linkage analysis service, N-glycans are released from glycoproteins by PNGase F reaction. The released N-glycan is then chemically labeled at a free reducing termini with fluorescent tags 2-aminobenzamide (2-AB). The labeled glycans are reacted sequentially with a series of exoglycosidases. The reaction products from each step of the reaction are analyzed by HILIC with a fluorescence and mass spectrometric detection.

In this procedure, the detailed structures of the glycans, including the configuration and position of glycosidic linkages, can be obtained based on the specificities of the exoglycosidases,  the HPLC separation profiles after each step of the sequential reactions, MS and MS/MS information of the reaction products at each step. Since the linkage information is based on several lines of evidence, the determination of glycan linkage is highly reliable.

3. Site-Specific N- or/and O-Glycosylation Analysis

Where the glycoprotein contains more than one  N-linked glycosylation site, released glycan analysis may not be able to provide glycan structures at each individual glycosylation site. To address this issue, we offer our site-specific glycosylation analysis to determine the glycosylation sites and glycan structures at each glycosylation site.

There is no routine enzymatic reaction that can completely remove O-linked glycans. Also, it is very common that O-glycosylation is present at multiple sites in a protein since there is no strict consensus site for O-glycosylation.  So site-specific glycosylation analysis is often carried out for analysis of the modification.  The procedure is similar to site specific N-glycosylation analysis and is often carried out with the same sample.

In this service, a glycoprotein sample is digested with several different proteolytic enzymes for complete coverage and analyzed by nano LC-ESI-MS/MS. MS and MS/MS data are analyzed by our proprietary software to identify all glycopeptides based on their MS/MS features. The amino acid sequence and predicted glycan structure are determined by the software and then validated manually with MS/MS spectra assignments. The glycopeptides with the same peptide sequence but different glycan structures are clustered together and the ratio of each glycoform is calculated based on EIC of the corresponding glycopeptides. 

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