Targeted N-Glycosylation Site Mapping Service

MtoZ Biolabs provides professional Targeted N-Glycosylation Site Mapping Services for precise identification, localization, and characterization of N-linked glycosylation sites in specific target proteins. Leveraging advanced LC-MS/MS platforms and optimized glycopeptide workflows, we deliver high-confidence, site-specific glycosylation data to support biopharmaceutical development, structural biology research, and quality control.

What Is N-Glycosylation?

N-Glycosylation is a key post-translational modification in which glycans are covalently attached to the nitrogen atom of asparagine residues within the consensus sequence Asn-X-Ser/Thr. This process occurs co-translationally in the endoplasmic reticulum and is further modified in the Golgi apparatus, producing a diverse array of glycan structures. It is typically classified into three major types: high-mannose, complex, and hybrid N-glycans. N-glycosylation plays essential roles in protein folding, stability, solubility, secretion, and receptor recognition.

Zlatina, K. L. et al. Biochem Cell Biol. 2021.

Figure 1. The Three General Types of N-Glycans

Targeted N-Glycosylation Site Mapping is the precise identification and localization of these glycan attachment sites on a specific protein. Unlike broad glycoproteomic profiling, this targeted approach focuses on defined proteins, often therapeutic antibodies, recombinant enzymes, or other biologics, to determine exactly which asparagine residues are glycosylated and what glycan structures are present at each site.

Targeted N-Glycosylation Site Mapping Service at MtoZ Biolabs

MtoZ Biolabs offers targeted N-glycosylation analysis tailored to client-defined proteins. Our service provides detailed, reproducible, and publication-ready data that enable confident structural and functional interpretation.

Our core offerings include:

💠N-Glycosylation Site Identification: Locate glycan attachment sites on target proteins using high-resolution LC-MS/MS analysis.

💠Site-Specific Glycan Profiling: Determine glycan composition (mannose, fucose, GlcNAc, galactose, sialic acid) and structural heterogeneity at each site.

💠Quantitative Occupancy Analysis: Measure site-specific glycan occupancy and relative abundance to monitor sample consistency.

💠Glycan Structural Characterization: Identify branching, linkage type, and terminal modifications for complete glycan structure elucidation.

Workflow of Targeted N-Glycosylation Site Mapping Service

MtoZ Biolabs applies a precise and validated workflow to identify N-glycosylation sites on target proteins through enzymatic digestion, isotopic labeling, chromatographic separation, and mass spectrometric detection.

1. Protein Preparation and Digestion

Purified or extracted proteins are first denatured and subjected to enzymatic digestion, typically with trypsin, to generate peptides suitable for downstream analysis.

2. Enzymatic Deglycosylation with PNGase F

Samples are treated with peptide N-glycosidase F (PNGase F) in heavy water (H218O). During this reaction, the attached N-glycan is released, and the glycosylated asparagine is converted into aspartic acid containing an 18O atom. This isotope incorporation increases the peptide's molecular weight, allowing clear differentiation between glycosylated and non-glycosylated forms.

3. Peptide Separation by LC

Following deglycosylation, peptides are separated using liquid chromatography (LC) to resolve complex peptide mixtures and enhance detection sensitivity. Retention time differences between modified and unmodified peptides aid in glycosylation site confirmation.

4. Mass Spectrometry Detection

High-resolution LC-MS/MS is then employed to analyze the peptides. The mass shift from 18O incorporation enables accurate localization of N-glycosylation sites. Fragmentation spectra provide both sequence verification and modification evidence.

5. Data Processing and Site Confirmation

Acquired spectra are processed using specialized bioinformatics tools to match peptides to the protein sequence, identify 18O-labeled sites, and confirm N-glycosylation positions with high confidence.

Why Choose MtoZ Biolabs

✔️High Sensitivity and Accuracy

Advanced LC-MS/MS platforms ensure precise detection of low-abundance glycopeptides and accurate localization of N-glycosylation sites.

✔️Reliable Quantification

Isotopic labeling technology enables confident differentiation and quantitative comparison of glycosylated versus non-glycosylated peptides.

✔️Customizable Analytical Solutions

MtoZ Biolabs designs tailored analytical workflows to fit specific protein targets, sample types, and research objectives.

✔️Reproducible and Standardized Workflow

Each experiment follows rigorously optimized protocols, ensuring consistent, high-quality results suitable for both academic and biopharmaceutical applications.

Applications of Targeted N-Glycosylation Site Mapping Service

1. Therapeutic Protein Characterization: Verify glycosylation profiles in antibodies, enzymes, and fusion proteins to ensure correct folding and stability.

2. Biosimilar Development: Compare N-glycosylation sites and structures between biosimilars and original biologics.

3. Quality Control and Process Optimization: Monitor glycosylation consistency during expression, purification, and storage.

4. Disease Mechanism Studies: Investigate site-specific glycosylation changes in disease-associated proteins.

FAQ

Q1: What Types of Samples Are Suitable?

A1: We accept a wide range of protein and biological samples, including purified proteins, recombinant proteins, antibodies, and other therapeutic proteins. Cell lysates, tissues, and biological fluids can also be submitted, with MtoZ Biolabs performing target protein extraction and preparation.

Q2: How Should I Prepare My Samples?

• All samples should be stored at –80°C.
• Use dry ice or ice packs during shipment to preserve protein integrity.
• Avoid freeze–thaw cycles that may alter disulfide linkages.

For more information, please refer to Sample Submission Guidelines for Proteomics.

Q3: What Is the Service General Workflow?

 

Q4: What Data Formats Are Provided?

Results are delivered in standard and easy-to-access formats, including:

• Raw MS data files (.raw, .wiff, .d)
• Processed data and quantification tables (.xlsx, .csv)
• Spectral and chromatogram images (.pdf, .png)
• Final analytical report (.pdf)

All deliverables are compatible with common data analysis software. Additional file formats or data structures can be provided upon request to meet specific research or publication requirements.

Start Your Project with MtoZ Biolabs

Contact us to discuss your experimental design or request a quote. MtoZ Biolabs provides high-quality Targeted N-Glycosylation Site Mapping Services to help clients accurately define glycan attachment sites, compositions, and structural variations on specific proteins.