N-Glycan Profiling Service

Creative Proteomics with expertise in analyzing glycosylation (N-Glycan Profiling and O-Glycan Profiling) from a variety of protein samples. We can conduct comprehensive analysis of N-glycan, including glycan composition, connection mode and site-specific information through advanced mass spectrometry technology and professional bioinformatics analysis platforms. Our professional team will provide customers with full support from sample preparation to result interpretation, helping you to deeply understand the role of glycosylation in biological functions and accelerate your research and development process.

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Techniques
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FAQ
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What Is N-Glycan Profiling?

N-glycosylation usually occurs at a specific motif in the protein sequence, namely Asn-X-Ser/Thr (X can be any amino acid except proline). N-Glycan Profiling is one of the most common types of post-translational modifications of proteins, analyzing the structure and composition of N-glycan in proteins, revealing the impact of glycosylation modifications on protein function, cell biology, and disease. As a critical quality attribute of biopharmaceuticals, N-Glycan profiling plays an import role in ensuring the quality and safety of biologics.

Our Advanced N-Glycan Profiling Service

We provide a variety of analytical methods for N-Glycan analysis, including but not limited to the mentioned below.

Glycan structure identification
Glycosylation site identification
Glycan quantification
Glycan sequence and linkage information
Glycan heterogeneity

General N-Glycan Profiling Workflow

The general procedure of N-glycan analysis:

1. Sample preparation

Protein sample is extracted from the target protein or protein mixtures. There will be purification and concentration steps of the protein to ensure the accuracy and efficiency of the N-glycan profiling analysis.

2. Release of N-glycan

The protein sample is treated with a specific enzyme (such as PNGase F) to release the covalently attached N-glycan.

3. Glycan purification

The released N-glycan may need to be further purified by chromatography or other methods to remove proteins, peptides, and other substances that may interfere with the analysis.

4. Labeling (optional)

To improve the sensitivity and resolution of detection, the purified N-glycan can be fluorescently labeled. (2-AB)

5. Separation and detection

N-glycans are separated and detected using techniques such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), or mass spectrometry (MS). Providing qualitative and quantitative information of the glycans.

6. Data analysis

Conduct in-depth analysis of the acquired data.

Techniques For N-glycan Profiling

MALDI-TOF-MS

Advantages: Suitable for high throughput detection and highly sensitive for the structural characterization of glycosylated compounds.

Commonly used to analyze N-glycan in some biological samples such as plasma, cells and tissues.

N-Glycan Profiling Service Figure 1. MALDI-TOF-MS results confirm the modulation of N-glycans from lactoferrin linked with MIE-NH₂ ^[1]

HILIC-UHPLC-MS

Advantages: Separation of glycans by hydrophilic interaction chromatography and detected by MS. Suitable for the analysis of a large number of samples and can provide detailed N-glycan structure information.

N-Glycan Profiling Service Figure 2. Overlaid total ion current chromatograms (TICCs) of RapiFluor-MS labelled IgG N-glycans from a patient with MOGS-CDG (red line) and control (black line) by HILIC-UPLC-ESI-MS ^[2]

CE-MS

Advantages: Suitable for analyzing low-abundance and similar N-glycans, and for resolving the heterogeneity of N-glycans with high resolution using minimal samples.

N-Glycan Profiling Service Figure 3. N-Glycan isomer differentiation by CE-MS ^[3]

What Samples We Analyze?

Biopharmaceuticals: Antibodies, peptide vaccines, Fc fusion proteins, etc.

Biological samples: Plasma, tissues, fluids, cell supernatant, lysate etc.

Other: Sialic acids, microbial polysaccharides, plant proteins, etc.

In addition to the samples listed above, we have rich experience in analyzing samples from a variety of sources, please contact us to discuss the feasibility of your samples.

FAQ

What are the applications of N-glycan profiling?

Biomedical research: Used for the development and quality control of biopharmaceuticals (such as therapeutic monoclonal antibodies).

Disease diagnosis and research: Some of the diseases affect the glycan pattern on the cell surface, N-glycan analysis plays an important role in the disease markers identification and mechanism research.

Cell biology and immunology: Analysis of changes in glycan on the cell surface helps understand cell-to-cell interactions and signal transduction.

How to choose a suitable protein sample?

Choose high-purity, high-concentration protein sample to avoid interference from impurities. For low-abundance protein, enrichment is recommended (such as immunoprecipitation).

Why do we need to label glycan?

Labeling glycan improves detection sensitivity (such as fluorescence or mass spectrometry detection)

What are the technical challenges of N-glycan profiling?

Complex structure: The glycan structure is complex, increasing the difficulty of analysis.

Low abundance and low ionization efficiency: Glycan is low abundance and low ionization efficiency in mass spectrometry, which affects the detection sensitivity.

Sample volume requirements: Traditional glycomics analysis usually requires a large amount of sample, which is a challenge for trace sample analysis.

Learn about other Q&A.

N-Glycan Profiling Case Study

Article

Case study: ldentification of low abundant isomeric N-glycan structures in biological therapeutics by LC/Ms

Publications

Here are some publications from our clients:

Conformational differences between functional human immunodeficiency virus envelope glycoprotein trimers and stabilized soluble trimers. 2019. https://doi.org/10.1128/jvi.01709-18
Identification of the O-Glycan Epitope Targeted by the Anti-Human Carcinoma Monoclonal Antibody (mAb) NEO-201. 2022. https://doi.org/10.3390/cancers14204999
Modulation of the Endomembrane System by the Anticancer Natural Product Superstolide/ZJ-101. 2023. https://doi.org/10.3390/ijms24119575
Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells. 2021. https://doi.org/10.1126/sciadv.abe8349

More Publications

References

Senan, A.M.; et al. Modification and characterization of lactoferrin-iron free with methylimidazolium N-ethylamine ionic liquid as potential drugs anti SARS-CoV-2. Engineering Proceedings. 2023, 37(1):14. https://doi.org/10.3390/ECP2023-14701
Messina A.; et al. HILIC-UPLC-MS for high throughput and isomeric N-glycan separation and characterization in congenital disorders glycosylation and human diseases. Glycoconjugate Journal. 2021, 38(2):201-211. https://doi.org/10.1007/s10719-020-09947-7
Wagt S.; et al. N-Glycan isomer differentiation by zero flow capillary electrophoresis coupled to mass spectrometry. Analytical Chemistry. 2022, 94(38):12954-12959. https://doi.org/10.1021/acs.analchem.2c02840

* For Research Use Only. Not for use in diagnostic procedures.

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From Our Clients

"I recently used their proteomics service for a project analyzing protein interactions in yeast models. The team was very responsive and helped clarify the methodology they employed, which made me feel confident in the results. The data quality was solid, with clear identification of several key proteins involved in our study. Their thorough analysis enabled me to pinpoint specific interactions that I hadn't considered before, which significantly improved the direction of my research. I appreciate their professionalism and support throughout the process."

Sarah Thompson, University of California, Berkeley

"Our lab collaborated with them on a project studying cancer biomarkers. The proteomics analysis provided was detailed and focused, specifically highlighting the differential expression of proteins between healthy and tumor samples. Their clear explanations of the data helped my team understand the biological implications. I also appreciated their willingness to revise the reports based on our feedback, ensuring that we had everything we needed for our publication. This collaborative spirit was invaluable."

Emily Rodriguez, Stanford University

"Our lab worked with them on a project studying the effects of diet on gut microbiota using proteomics. They used a label-free quantification method to analyze proteins in fecal samples before and after dietary intervention. The results showed significant changes in protein expression linked to microbial activity. This was pivotal for our hypothesis about diet-microbiota interactions. The clarity of their data presentation made it easy for our team to integrate these findings into our ongoing research."

Dr. Lisa Wong, University of Toronto

"My experience with Creative Proteomics during the mass spectrometry analysis was excellent. We sent in human saliva and mouse brain tissue samples, which they expertly analyzed using both LC-MS and GC-MS techniques. The results were invaluable, revealing key metabolites in the saliva and identifying biomarkers linked to brain function in the brain tissue."

Dr. Emily Carter, Senior Research Scientist

"The overall service from Creative Proteomics was outstanding. They made the entire process seamless and efficient, allowing us to focus on our research. We worked with leaf and root samples from various Arabidopsis genotypes for targeted metabolomics analysis. Their thorough profiling of primary and secondary metabolites gave us important insights into how the plants respond metabolically to environmental stress."

Dr. Laura Henderson, Plant Physiologist

"We had a pleasant collaboration with Creative Proteomics on mass spectrometry analysis of lipids. They conducted a detailed analysis of lipid species, providing us with important insights into lipid metabolism and its relationship with metabolic syndrome disease states."

Dr. Sarah Mitchell, Research Scientist

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