N-glycans are linked to proteins through the carboxamide function of an asparagine (Asn) residue in the consensus sequence Asn-X-Ser/Thr (X ≠ Pro).
For their analysis, N-glycans are enzymatically released from glycoproteins with PNGase F for mammalian and PNGase A for N-glycan structures with core alpha 1,3 fucose residues (plant, parasite and insect structures), often labelled or chemically stabilized and analysed by different Mass Spectrometry-based techniques, depending on your needs:
MALDI-TOF profiling of released N-glycans
Glycans can be permethylated to improve ionization efficiency and to stabilize sialic acid residues. It is possible to achieve absolute quantification by spiking the sample with our stable isotope-labeled (SIL) glycan standards (CarboQuant). An additional sialic acid derivatization, which differentiates between α-2,3 and α-2,6 linkages, can be applied before analysis.
UHPLC-FLD-MS profiling of released N-glycans
Provides a quantitative profile of all N-glycans present in a sample based on their mass and retention time. This method employs ultra-high-performance liquid chromatography (UHPLC) to separate the fluorescently labelled glycans, including most isomers.
Before analysis, N-Glycans are derivatized with a fluorophore like 2-AB, 2-AA, RapiFluor, or procainamide, which improves the chromatographic separation of the glycan mixture and permits the relative quantification of all glycans by fluorescence detection. Our advanced MS-quantification protocol allows for the rapid identification and quantification of glycans by mass spectrometry in case of peak overlap or for high-throughput applications with reduced UPLC run time.
UHPLC-MS analysis of N-glycan released from mammal tissue (80-120 N-glycans quantified)
How we work for N-Glycan Profiling
Our workflow ensures a clear, straightforward, and transparent process for sample glycoanalysis
Sample
Let us know which type of sample (purified protein, antibodies, cells, tissue, etc.) you want to analyze, and we will apply the most convenient sample preparation protocol.
Glycan release and isolation
We will use the optimal enzymes, techniques, and procedures to have the glycans ready to be analyzed.
Chemical labelling of glycans
We will choose the labeling method according to the detection techniques.
Sample purification
We enrich and purify the samples to obtain the best signals.
Mass spectrometry analysis
The latest technology and instruments are used to ensure the data quality.
Data processing
Our experts analyze and interpret the data to transform it into actionable information.
Report
A publication-type report that includes the Materials & Methods, data interpretation, and potential further analysis.
Purposes and applications for N-Glycan Profiling
N-glycans of a purified glycoprotein or more complex biological matrices serve several purposes and applications:
Biotechnology
- Monitoring Glycan profile of recombinant antibodies and therapeutic proteins during clone selection, scale-up, change of expression system, and manufacturing according to EMEA guidelines
- Test for presence of immunogenic glycan features (Galili-antigen, Neu5Gc)
- Optimization of glycan profile of biosimilars
Biomarkers & Diagnostics
Disease-specific N-glycan alterations serve as sensitive biomarkers, improving early diagnosis, patient stratification, and monitoring of therapeutic response
Changes in glycan profiles (whole serum, IgG, single protein markers,..) are very sensitive to disease onset and progression. Asparia provides support for study design, statistical analysis, and biomarker validation
Infectious Diseases
In infectious diseases, complex and altered N-glycans are exploited by pathogens for cell entry; profiling these interactions informs vaccine design and antiviral glycomimetics
Oncology
N-glycan profiling reveals tumor-associated glycosylation changes that drive metastasis and immune escape, enabling biomarker discovery and the development of glycomimetic cancer therapeutics
Immune & Inflammatory Diseases
N-glycan signatures on antibodies and receptors modulate immune activation; their characterization guides diagnostics and the design of glycoengineered therapies
Microbiome
Microbial enzymes modify host N-glycans, shaping symbiosis and dysbiosis; profiling supports prebiotic strategies and next-generation microbiota-based interventions
Other Fields
- Veterinary: N-glycan biomarkers aid in disease diagnosis and the development of vaccines for livestock and companion animals
- Plants and Agriculture: N-glycan analysis uncovers stress-resistance mechanisms
- Nutraceuticals and Cosmetics: it supports glycan-based bioactive design
- Human Milk Oligosaccharides (HMOs): It guides the development of glycan mimetics for infant nutrition and immune protection
Related publications
A selection of our most relevant scientific publications that form our N-Glycan Profiling analysis
Álvarez J, Parody N, Calzada D, Aranda T, Renshaw A, Serna S, et al. Correlation between N-glycan GnGnXF3 and the allergic immune response against Juniperus ashei pollen. Allergy. 2025.
Serna S, Comino N, Reichardt NC, López-Gallego F. Preparative isolation of N-glycans from natural sources mediated by a deglycosylating heterogeneous biocatalyst in flow. ChemSusChem. 2025;18(10): e202402346.
Alvarez J, Aranda T, Ruiz R, Aramendia L, Reichardt N-C, Carnes J. N-glycans other than MUXF3 are targets for specific IgE antibodies against Juniperus ashei pollen allergens. J Allergy Clin Immunol. 2023;151(2):AB212.
Lossio CF, Osterne VJS, Pinto-Junior VR, Chen S, Oliveira MV, Verduijn J, et al. Structural analysis and characterization of an antiproliferative lectin from Canavalia villosa seeds. Int J Mol Sci. 2023;24(21):15966.
Clos-Sansalvador M, Garcia SG, Morón-Font M, Williams C, Reichardt N-C, Falcón-Pérez JM, et al. N-glycans in immortalized mesenchymal stromal cell-derived extracellular vesicles are critical for EV-cell interaction and functional activation of endothelial cells. Int J Mol Sci. 2022;23(17):9539.
