O-Glycan Profiling Services

O-Glycosylation is a critical form of protein post-translational modification, attaching O-glycans primarily to serine (Ser) or threonine (Thr) residues to regulate protein folding, stability, and biological activity. Unlike N-glycosylation, O-glycosylation lacks conserved core structures and exhibits remarkable structural diversity, including mucin-type O-GalNAc, nuclear-regulatory O-GlcNAc, and Notch-related O-fucose modifications. These O-glycans play vital roles in cell adhesion, immune responses, and disease processes such as cancer metastasis and inflammatory bowel disease (IBD).

 



Figure 1. Depiction of the N-Glycan and O-Glycan Core Structure

 

Aberrant O-glycan expression is closely associated with many diseases. For instance, truncated mucin-type O-glycans mark tumor immune evasion, and dynamic changes in O-GlcNAc modifications influence neurodegenerative disease progression. However, the structural complexity and heterogeneity (e.g., short-chain modifications, isomers) pose significant analytical challenges. MtoZ Biolabs offers O-Glycan Profiling Services that provide comprehensive solutions from structural characterization to functional correlation by employing cutting-edge mass spectrometry and customized analytical strategies to make breakthroughs in disease research and translational medicine.

 

The service can decipher the complete landscape of O-glycans in your biological samples, enabling researchers to:  

1. Precisely Characterize O-Glycan Composition and Structures: Identify core structures such as O-GalNAc, O-GlcNAc, and terminal modifications including sialic acid and fucose.

2. Quantify Differential Expression Profiles: Capture abundance variations between disease and control groups to pinpoint potential biomarkers.

3. Resolve Structural Isomers: Distinguish isomers to reveal fine structural dynamics and functional relevance.

4. Analyze Diverse Sample Types: Compatible with cells, tissues, biofluids (serum, saliva, cerebrospinal fluid), and recombinant proteins.

 

Analysis Workflow  

1. Sample Preparation and Glycan Release  

• Gentle Chemical Release: Optimized β-elimination minimizes "peeling" effects to preserve native glycan structures.
• Reducing-End Stabilization: Chemical derivatization at the reducing end improves stability and detection sensitivity.

 

3. High-Resolution Separation and Mass Spectrometry  

• Multidimensional Chromatography: Combining Hydrophilic Interaction Chromatography (HILIC) and reversed-phase chromatography (C18) enhances separation efficiency for polar glycans and structural isomers.
• Ion Mobility-Mass Spectrometry (IM-MS): Utilizes collision cross-section (CCS) measurements for accurate isomer differentiation.
• Tandem Mass Spectrometry (MS/MS): High-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD) generate diagnostic fragment ions, pinpointing glycosylation sites.

 

4. Data Interpretation and Reporting  

• Automated Glycan Annotation: High-throughput O-glycan annotation through fragment ion matching and spectral library comparisons.
• Comprehensive Reporting: Delivers glycan composition tables, differential expression heatmaps, and biological insights.

 



Figure 2. The Workflow of O-Glycan Profiling

 

Why Choose MtoZ Biolabs? 

1. Exceptional Sensitivity and Resolution  

Our O-Glycan Profiling Services detect glycan structures at femtomole (fmol) levels, ideal for low-abundance samples (e.g., clinical biopsies) and structural isomers.

 

2. Customized Analytical Solutions  

Tailored sample preparation protocols according to sample complexity (e.g., mucus, exosomes).

 

3. Rapid Turnaround and Full Technical Support  

•  Standard delivery within 10-14 business days; expedited services available upon consultation.
•  One-on-one technical consultation covering experimental design, analytical support, and data interpretation.

 

Applications  

1. Tumor Microenvironment Studies  

Characterizing mucin-type O-glycans on tumor cells to explore correlations with immune checkpoint inhibitor responses.

 

2. Inflammatory Disease Mechanisms  

Investigating alterations in sulfation/sialylation ratios of mucin O-glycans in COPD and IBD to illuminate barrier dysfunction mechanisms.

 

3. Neurodegenerative Biomarker Discovery  

Measuring O-GlcNAc modification levels in cerebrospinal fluid to investigate links with Alzheimer’s and Parkinson’s diseases.

 

4. Biotherapeutic Quality Control  

Evaluating batch-to-batch consistency of therapeutic antibodies through O-glycosylation profiling to ensure product stability and efficacy.

 

Case Study

1. Ion Mobility-Tandem Mass Spectrometry of Mucin-Type O-Glycans

This study describes a rapid ion mobility-tandem mass spectrometry (IM-MS/MS) approach for analyzing mucin-type O-glycans. Unlike conventional liquid chromatography-mass spectrometry (LC-MS), which suffers from lengthy analysis time and poor reproducibility when separating O-glycan isomers, the IM-MS/MS technique efficiently resolves complex O-glycans, reducing analysis time from one hour to two minutes. The method was successfully applied to sputum samples from cystic fibrosis patients, enabling rapid and accurate identification of disease-associated O-glycosylation features. O-Glycan Profiling Services utilize advanced ion mobility-tandem mass spectrometry (IM-MS/MS) for rapid and efficient separation and identification of mucin-type O-glycans. By quickly analyzing complex biological samples, the service can greatly enhance analytical efficiency and precisely characterize O-glycan structures and isomer distributions associated with diseases.

 



Bechtella, L. et al. Nat Commun. 2024.

Figure 3. TIM-MS of Deprotonated O-Glycans from Porcine Gastric Mucins

 

2. In-Depth Profiling of O‑Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography−Mass Spectrometry and Glycogenomics

This study developed a high-throughput analytical approach for detailed profiling of O-glycan isomers in human cells, employing nonreductive glycan release and reducing-end labeling coupled with C18 nanoliquid chromatography and Orbitrap mass spectrometry. Utilizing genetically glycoengineered cell libraries, researchers successfully identified various complex O-glycan structures in human keratinocytes, including O-fucose, O-glucose, O-GlcNAc, and elongated core1 and core2 O-GalNAc glycans, demonstrating the method's potential for glycomic studies using standard analytical instruments. O-Glycan Profiling Services employ nonreductive glycan release and reducing-end labeling techniques, combined with efficient C18 nanoliquid chromatography and Orbitrap mass spectrometry, enabling high-throughput separation and identification of O-glycan isomers in complex samples, and providing comprehensive structural characterization of O-glycans in biological materials.

 



De, Haan, N. et al. Anal Chem. 2022.

Figure 4. Structural Characterization of Two Elongated O-GalNAc Core2 Isomers

 

Deliverables  

• Qualitative O-glycan composition analysis (glycan types and relative abundances).
• Isomer resolution, differential glycan screening, and KEGG/GO enrichment analyses.
• Glycosylation site identification (for glycoprotein-rich samples).

 

O-glycosylation is the hidden language of life regulation, whose complexity demands systematic exploration. MtoZ Biolabs’ O-Glycan Profiling Services, with unmatched technical capability, analytical accuracy, and flexible service offerings, aim to become your trusted partner in exploring disease mechanisms and developing novel therapies. Whether you are conducting basic research or clinical translation, we look forward to collaborating with you to unravel the secrets of O-glycans and expand the frontiers of precision medicine. Contact our technical team today to customize your O-glycan profiling solutions!