The technique is used to determine whether N-linked glycans occupy specific asparagine (Asn) residues in a protein. Although a protein may have multiple potential N-glycosylation sites (based on the consensus sequences NX-S/T), not all are necessarily glycosylated, and some may be only partially occupied.
This analysis involves proteolytic digestion (e.g., with trypsin or endo GluC), followed by treatment with PNGase F in 18O labeled water. During this reaction, the glycosylated asparagine residues are deamidated to aspartic acid with incorporation of an 18O atom, producing a mass change detectable by mass spectrometry. The use of 18O water allows discrimination between a true glycosylated site and an artifactual asparagine deamidation. Site occupancy at a site is calculated from the ratio of the 18O deamidated peptide to the native peptide (N).
We can perform the analysis on a broad range of samples, including recombinant glycoproteins, monoclonal antibodies, or endogenous proteins from tissues, cells, or serum. The sequence of the protein to analyze is required for the analysis, and we normally work with 100 – 200 μg of purified protein.
How we work for N-glycosylation Site Occupation Analysis
Our workflow ensures a clear, straightforward, and transparent process for sample glycoanalysis
Sample
Purified protein (recombinant antibodies, glycoproteins, endogenous proteins from cells, tissues…) should be our starting material (100 – 200 μg). Let us know if we can help to get the purified material.
Protease Digestion
Depending on your protein sequence, we perform a customized protease digestion.
Removal of N-glycans
We perform an enzymatic removal of the N-glycans in 18O labeled water.
Sample purification
We enrich and purify the samples accordingly 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-glycosylation Site Occupation Analysis
Oncology
This analysis helps uncover glycosylation changes in tumor-associated proteins, which often affect how cancer cells grow, invade, or evade the immune system. The technique can identify partially glycosylated or abnormally glycosylated sites in biomarkers or therapeutic targets, aiding in biomarker discovery and drug design.
Infectious Diseases
Pathogens often produce glycoproteins with specific glycosylation patterns. Analyzing site occupation helps assess how viral envelope proteins (e.g., HIV gp120 or SARS-CoV-2 spike protein) are glycosylated, which is crucial for vaccine development and understanding immune evasion mechanisms.
Immune and Inflammatory Diseases
As site occupancy can influence antibody function, such as effector activity or half-life, it’s particularly important in therapeutic antibody engineering to ensure correct glycosylation profiles through this technique.
Microbiome
Site occupation analysis can be used to study host proteins modified by microbial activity or microbial glycoproteins themselves, helping to explore how glycosylation patterns are shaped by or interact with gut microbes.
Biomarkers & Diagnostics
Many serum proteins used as biomarkers are glycosylated, and site-specific occupation can change in disease states. This technique allows researchers to detect incomplete or altered glycosylation, providing insights into conditions like cancer, liver disease, or congenital glycosylation disorders.
Other fields
- Plant biology and agriculture: it supports the study of defense proteins, enzymes, and allergens, helping optimize genetically modified crops or plant-based biopharmaceuticals.
- Nutraceutical and Cosmetics industries: it ensures the consistency and activity of glycoproteins used in functional foods and skincare products. For Human Milk Oligosaccharides (HMOs), it helps confirm proper glycosylation of added bioactives, ensuring product safety and efficacy.
