N-glycan profiling has been widely used in the identification of various diseases, including cancer, Alzheimer's, and diabetes. CD BioGlyco has rich experience in glycan profiling. Our scientists use advanced lab equipment to process samples and generate results. CD BioGlyco has confidence to be your essential research assistant at every moment.

Background

Protein post-translational modification (PTM) has been at the center of research for the worldwide scientific community. The most common PTM of protein is glycosylation, especially N-glycosylation. The synthesis of N-linked glycan starts from the endoplasmic reticulum and completes in the Golgi apparatus. N-glycosylation modification plays an important role in the process of protein folding and transportation. Its occurrence will lead to changes in stability, solubility and conformation of the protein. As glycoproteins, cytokines, monoclonal antibodies and other therapeutic proteins become popular in clinical research, there is an increasing demand for N-glycosylation analysis of these objects. However, the glycosylation analysis process is challenging due to the complexity and isomerism of glycans.

Fig 1. The biosynthesis of N-linked glycans present in the antibody (Q, Zhou.; H, Qiu. 2019)

Services

At CD BioGlyco, we perform N-Glycan profiling use the following methods.

• N-Glycan profiling by HILIC-UHPLC MS

Hydrophilic interaction chromatography is a chromatographic technique used to improve the retention of strongly polar substances that are poorly retained in reversed-phase chromatography. InstantPC (ProZyme) and RapiFluor-MS (Waters) are used to label fluorophore to the glycosamine which produced after PNGase F digestion immediately and improve the sensitivity of the entire reaction. Before fluorescent labeling, detergents, non-volatile salts or substances with free amino groups which could interfere with the experiment should be removed.

• Excellent retention performance and strong separation ability
• No need to add ion pair reagent
• Advanced technique platforms
• High sensitivity

• N-Glycan profiling by MALDI-TOF-MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a new type of ionization biological mass spectrometry developed in recent years. It is suitable for high-throughput screening of proteins or other biological macromolecules and analyze the single nucleotide polymorphisms of oligonucleotides or genes. It is usually used as a powerful tool to analyze the molecular weight and molecular weight distribution of natural and synthetic polymers.

• Wide quality detection range and high-quality accuracy
• Fast analysis speed, strong molecular/ion peak, and intuitive information
• Strong tolerance for interference

Applications

• Proteins structure analysis
• Proteins high-throughput screening 
• Therapeutic proteins development

Advantages of Us

• Highly precision equipment
• Experienced researchers
• Highly reliable results
• Stability and consistency
• Cost-effective

Case Study

Background

Aberrant glycosylation, particularly within the N-glycan landscape, is a well-established hallmark of oncogenesis and cancer progression. Mapping the precise alterations in N-glycan composition, branching patterns, and fucosylation/sialylation traits unlocks vital functional biomarkers for early diagnosis and therapeutic monitoring. However, clinical cancer tissues and biological fluids are extraordinarily complex. The co-existence of dense O-glycoproteins often interferes with N-glycan isolation, and the sheer structural diversity of carbohydrate isomers demands an analytical platform capable of complete depolymerization, ultra-high-resolution separation, and sophisticated bioinformatic deconvolution to capture subtle, cancer-specific glycan features.

Our Solution

CD BioGlyco established a specialized, multi-dimensional enzymatic N-glycan release and orthogonal MS characterization solution to perform deep-scale profiling of samples.

• Targeted N-Glycan Liberation & Partitioning: Samples were fully homogenized using an SDS-sonication matrix and reduced with 1,4-dithiothreitol (DTT) to expose buried glyco-epitopes. Following desalination, N-glycans were cleanly and selectively cleaved from the peptide backbone using peptide N-glycosidase F (PNGase F) overnight. Utilizing an advanced column chromatographic system, the liberated N-glycans were separated with high fidelity from the remaining intact O-glycoprotein fraction, ensuring an interference-free target pool.
• High-Performance Orbitrap Characterization: The isolated native N-glycan fractions were directly analyzed on a state-of-the-art MS operating in negative ion mode, which provides unmatched mass accuracy and exceptional sensitivity for complex branched and acidic/sialylated glycoforms.
• Advanced Deconvolution & Feature Mapping: The complex multi-charge raw mass spectra were processed to translate raw data into clean, single-charge neutral mass profiles.

Results

The optimized N-glycan profiling solution provided the client with an exhaustive, publication-ready data package that successfully unlocked the sample's glycan signature:

• Deconvoluted ESI-MS Fingerprinting: High-resolution mass spectra successfully generated fully deconvoluted profiles of the cancer-derived N-glycans, providing accurate mass measurements for unambiguous composition assignment.

Fig.2 Deconvoluted ESI-MS of sample N-glycans. (CD BioGlyco)

Table 1 Result of N-glycan identified. (CD BioGlyco)

• Cancer-Specific Feature Distribution: Specialized data processing resolved the exact distribution of crucial glycan features, including total fucosylation, core/antennary modifications, and sialylation levels, establishing a clear comparison matrix against baseline controls.

Table 2 N-glycan feature distribution. (CD BioGlyco)

• Deep Architectural Interrogation: By implementing an in-house bioinformatic filter that excluded dominant, non-specific oligomannose compositions, our team successfully unmasked the low-abundance, complex multi-antennary and sialylated N-glycan features. This critical step allowed the client to pinpoint subtle, statistically significant alterations driving cancer-associated phenotypes.

CD BioGlyco provides a full range of glycomics analysis services, including glycan analysis, site occupation, linkage analysis, glycan sequencing, etc, as well as many customized services. We use professionalism and efficiency to complete customer’s orders and save time and funds for customers while obtaining credible results.

Customers can contact our employees directly and we will respond promptly. If you are interested in our services, please contact us for more detailed information.

Reference:

1. Q, Zhou.; H, Qiu. The Mechanistic impact of N-Glycosylation on stability, pharmacokinetics, and immunogenicity of therapeutic proteins. Journal of Pharmaceutical Sciences. 2019, 108:1366-1377.

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