O-glycans are linked to proteins through the hydroxyl oxygen of serine (Ser) or threonine (Thr) residues.
For their analysis, O-glycans are chemically released by beta elimination with a strong base, followed by direct reduction to the stable alditol or by labeling with a fluorescent tag for chromatographic separation, before analysis by MALDI-TOF MS or UPLC-FLD-MS.
MALDI-TOF profiling of released O-glycans
Glycans can be permethylated to improve ionization efficiency and to stabilize sialic acid residues; a more specific sialic acid derivatization can be applied to differentiate α-2,3 and α-2,6 linkages before analysis. Permethylation also increases the mass of O-glycans to avoid interference with the region of matrix ions (up to 600 Da). It is possible to achieve absolute glycan quantification by spiking the sample with our stable isotope-labeled (SIL) glycan standards (CarboQuant).
MALDI-TOF profile of permethylated O-glycans from bovine maxillary gland mucin
UHPLC-FLD-MS profiling of released O-glycans
Provides a quantitative profile of all O-glycans present in a sample based on their mass and retention time. After chemical cleavage with a strong base, the released glycans can be either stabilized by reduction to their alditols or further 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.
Glycans can be identified by their mass, retention time, and fragmentation pattern generated by MS/MS methods available to our customers. In addition, the analysis of glycan mixtures after treatment with exoglycosidases, which are highly specific for the monosaccharide and linkage present, can help establish the exact nature in the case of ambiguous glycan structures.
UHPLC-MS analysis of permethylated O-glycans from bovine maxillary gland mucin
Detailed structural characterization via UHPLC-MS/MS analysis of permethylated O-glycans from bovine maxillary gland mucin
How we work for O-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 O-Glycan Profiling
O-glycans of a purified glycoprotein or more complex biological matrices serve several purposes and applications:
Oncology
O-glycan profiling identifies tumor-specific mucin-type glycosylation changes that influence invasion and metastasis, supporting biomarker discovery and glycomimetic-based cancer therapies
Biopharmaceutical development
- Characterization of therapeutic glycoproteins
- Biosimilar comparability studies, ensuring O-glycosylation patterns match reference products
- Quality control and batch consistency for recombinant proteins and monoclonal antibodies with O-glycosylation (e.g., hinge-region O-glycans)
- O-glycan analysis of (Gly₄Ser)₃ linker in fusion proteins
Biomarkers & Diagnostics
- Tumor-associated truncated O-glycans as potential cancer biomarkers
- O-glycan signatures as diagnostic, prognostic, or therapeutic response biomarkers, e.g., in cancer or autoimmune diseases
Immune and inflammatory diseases
Aberrant O-glycosylation of immune receptors and antibodies modulates inflammation; profiling enables diagnostic markers and the design of glycoengineered immunotherapies
Infectious Diseases
Pathogen-host interactions: O-glycans on mucins and cell surfaces mediate pathogen adhesion; profiling these interactions guides vaccine development and anti-adhesive therapeutics
O-glycan shielding on viral glycoproteins can affect immune evasion and vaccine design
Microbiome
O-glycans in the gut mucosa shape microbial colonization and host–microbe interactions; their analysis supports probiotic strategies and microbiota-targeted interventions
Other Fields
- Veterinary medicine: O-glycan biomarkers aid in animal disease monitoring and vaccine innovation
- Plant Biology and Agriculture: O-glycan analysis reveals stress and signaling mechanisms
- Nutraceuticals and Cosmetics: O-glycan profiling supports bioactive glycan discovery
- Human Milk Oligosaccharides (HMOs): O-glycan profiling assists the development of glycan mimetics for infant health
