Glycosylation Site Analysis Services

Glycosylation site analysis is to analyze the specific serine (Ser), threonine (Thr), or asparagine (Asn) residue on proteins where sugar chains are covalently attached. By precisely identifying glycosylation sites, glycosylation site analysis offers information about protein stability, activity, and molecular interactions. In biopharmaceutical development, glycosylation site heterogeneity can lead to variations in drug efficacy or immunogenicity risks, whereas in disease research, abnormal glycosylation sites serve as key biomarkers for cancer, neurodegenerative diseases, and other disorders.  

 

MtoZ Biolabs provides state-of-the-art mass spectrometry-based Glycosylation Site Analysis Services, integrating advanced sample preparation workflow to deliver high-sensitivity and high-coverage glycosylation site identification and heterogeneity analysis. Our service supports precision drug development, disease mechanism research, and biomarker discovery.  

 

MtoZ Biolabs’ Glycosylation Site Analysis Services include the following core aspects:  

1. Identification of N-Glycosylation and O-Glycosylation Sites  

Precisely mapping glycan attachment sites on serine (Ser), threonine (Thr), or asparagine (Asn) residues.  

2. Site-Specific Heterogeneity Analysis  

Quantifying the distribution of different glycoforms at the same site, including core fucosylation and sialic acid modifications.  

 

Analysis Workflow  

1. Sample Preparation and Enrichment  

• Extraction and enzymatic digestion (e.g., trypsin digestion) of glycoproteins from cells, tissues, or purified protein samples.  
• Targeted glycopeptide enrichment using oxidation-coupling methods or lectin affinity chromatography, improving detection sensitivity for low-abundance glycopeptides.  

 

2. Mass Spectrometry (LC-MS/MS) Analysis  

• High-resolution mass spectrometry (Orbitrap series) combined with electron transfer dissociation (ETD) to preserve glycan and peptide fragment information.  
• Optimized liquid chromatography gradients for effective separation of complex glycopeptides.  

 

3. Data Analysis and Validation  

• Localization of glycosylation sites based on glycopeptide-specific fragment ions.  
• Dual validation of site authenticity using deglycosylation enzymes (e.g., PNGase F), which introduces an aspartic acid conversion marker.  

 

4. Comprehensive Report Delivery  

• Glycosylation site lists with modification probability scores.  
• Heterogeneity heatmaps visualizing glycoform distributions.  
• Functional correlation analysis, such as predicting glycoform-dependent receptor binding activity.  

 



Oh, MJ. et al. Mass Spectrom Rev. 2025.

Figure 1. Streamlined Workflow for Site‐Specific Glycopeptide Analysis of Therapeutic Glycoproteins

 

Why Choose MtoZ Biolabs?  

1. Unmatched Sensitivity  

Our platform detects glycopeptides down to the fmol level, enabling reliable analysis of minute sample amounts.  

 

2. Broad Sample Compatibility  

We accept a variety of sample types, including cell lysates, serum, monoclonal antibodies, and recombinant proteins, accommodating complex biological matrices.  

 

3. Technical & Expertise Leadership  

• MtoZ Biolabs has completed over 1,000 glycosylation analysis projects, covering monoclonal antibodies, vaccines, and cell therapy drugs.  
• Our team has deep expertise in glycoprotein heterogeneity analysis and functional correlation interpretation.  

 

4. Fast Turnaround Time  

Standard workflow ensures full data delivery within 15 working days from sample submission to final report.  

 

Applications  

1. Biopharmaceutical Development  

Glycosylation site analysis is important in biopharmaceutical industry. It monitors glycosylation quality of antibodies and fusion proteins.   

 

2. Disease Mechanism Research  

• Identification of abnormal glycosylation sites in cancer and neurodegenerative diseases.  
• Discovery of glycosylation targets in pathogen-host interactions.  

 

3. Fundamental Research Support  

• Validation of glycosylation dynamics following gene editing or drug treatment.  
• Functional studies of glycosylation enzymes.  

 

Case Study

1. Site-Specific Profiling of Serum Glycoproteins Using N-Linked Glycan and Glycosite Analysis Revealing Atypical N-Glycosylation Sites on Albumin and α-1B-Glycoprotein 

This study employed a newly developed N-glycoproteomic method (NGAG) combined with LC-MS/MS and GPQuest software for site-specific profiling of serum N-glycoproteins. The results revealed that more than half of the identified N-glycosylation sites were modified by at least two glycans, with the majority being sialylated. Specifically, 3/4 of glycosylation sites were modified by biantennary N-glycans, and 1/3 were modified by triantennary sialylated N-glycans. Additionally, two novel atypical glycosylation sites (with an N-X-V motif) were identified and validated on albumin and α-1B-glycoprotein, with their widespread presence further confirmed through individual serum analyses. Glycosylation Site Analysis Services integrate high-resolution LC-MS/MS with bioinformatics tools to provide comprehensive N-glycosylation site analysis, revealing glycosylation heterogeneity and glycan distribution. By enabling site-specific glycosylation detection, it identifies various glycan modifications and uncovers potential atypical glycosylation sites, facilitating a deeper understanding of protein glycosylation characteristics.

 



Sun, S. et al. Anal Chem. 2018.

Figure 2. Identification and Validation of an Atypical N-Glycosite on Human Serum Albumin Using Sitespecific Glycosylation Analysis

 

2. Enhanced N-Glycosylation Site Analysis of Sialoglycopeptides by Strong Cation Exchange Prefractionation Applied to Platelet Plasma Membranes

This study presents a novel strategy for N-glycosylation site analysis, specifically applied to platelet membrane proteins. The approach integrates aqueous two-phase partitioning for membrane enrichment with a single-step strong cation exchange (SCX)-based purification of glycopeptides, leading to the identification of 148 glycosylation sites across 79 proteins. Notably, 69% of these sites were previously unannotated in the Swiss-Prot database, and 75% of the analyzed proteins were plasma membrane-localized. The method also supports miniaturization and relative quantification, demonstrating its potential application in broader proteomics studies, including those related to bleeding disorders, inflammation, and immune responses.  Glycosylation Site Analysis Services utilize advanced sample enrichment techniques and high-resolution LC-MS/MS for precise glycosylation site identification. By integrating efficient fractionation strategies, it enables comprehensive profiling of glycopeptides, particularly for membrane-associated proteins. The approach ensures high sensitivity in detecting unannotated glycosylation sites and supports quantitative analysis, making it applicable to various proteomics research areas.

 



Lewandrowski, U. et al. Mol Cell Proteomics. 2007.

Figure 3. Enhanced Fragment Ion Scans for Identification of Glycosylation Sites

 

Deliverables  

1. Comprehensive glycosylation site lists with a confidence level ≥95%. 

2. Quantitative comparison of glycosylation modifications under different conditions

3. Glycosylation heterogeneity distribution maps

4. Cross-sample differential analysis (PCA clustering and glycosylation network models) 

5. In-depth bioinformatics annotation (KEGG/GO functional enrichment analysis)  

 

Accurate glycosylation site analysis is key to decoding protein function and disease mechanisms. MtoZ Biolabs, with its advanced technological expertise and extensive project experience, is committed to delivering high-quality and reliable Glycosylation Site Analysis Services. Whether you are advancing biopharmaceutical clinical applications, exploring novel disease biomarkers, or uncovering fundamental biological insights, we provide data-driven solutions to accelerate your research and innovation. Submit your inquiry now and get a customized solution with expert consultation!