O-glycan profiling is extremely important for biological macromolecules and cell research. CD BioGlyco has rich experience in glycan profiling. Our knowledgeable scientists use the most advanced experimental equipment to process samples and analyze data. CD BioGlyco has confidence to be your essential research assistant in the field of glycobiology.

Background

Glycosylation is the process of adding carbohydrates to proteins or lipids under the control of enzymes. As a result of glycosylation, different proteins are marked with different labels, which change the conformation of peptides and increase the stability of proteins. Glycosylation can be categorized into 2 main types: N-linked and O-linked glycosylation. O-linked glycosylation is the transfer of carbohydrate chains to an oxygen atom of the hydroxyl group of serine, threonine, hydroxyproline or hydroxylysine. It is catalyzed by different glycosyltransferases and one monosaccharide is added each time. As with N-linked glycosylation, the last step is to add sialic acid residues. This reaction occurs in the trans face of the Golgi capsule and trans-Golgi network. In contrast to N-linked glycans, the release of O-linked glycans is a complex process due to the highly diverse nature of the O-glycan structure as well as the absence of specific enzymes that release the O-linked glycans attached to protein. Chemical release is the major method for O-linked glycans separation.

Fig 1. Schematic representation of structural elements in mucin type O-glycans (Li, Z.; Cai, W.G.2019)

Services

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

Releasing glycans from glycoproteins for the purpose of further characterization is the most commonly used method for analysis of protein glycosylation. The only enzymatic method (O-glycanase) and several chemical methods (Reductive β-elimination/ Non-reductive β-elimination) were developed over the past decades for releasing O-glycans. Then multiple comprehensive strategies based on MS are used to analyze after releasing.

Fig 2. O-Glycan profiling flow chart - CD BioGlyco

Applications

• Proteins and other biomacromolecules structure & function research
• Proteins high-throughput screening 
• Disease pathology and progression research
• Therapeutic proteins development

Case Study

Background

Unlike N-glycans, O-glycans cannot be cleaved by a universal enzyme, and they often lack a conserved core structure, leading to immense microheterogeneity. Furthermore, the co-existence of abundant N-glycans often masks the signals of lower-abundance O-glycoforms. To achieve precise O-glycan profiling, the solution effectively eliminates N-glycan interference and employs harsh yet controlled chemical release methods without causing sample degradation (peeling).

Our Solution

CD BioGlyco deployed an integrated enzymatic-chemical partitioning and high-sensitivity MALDI-TOF-MS analytical method to deliver an uncompromised structural blueprint of the O-glycan landscape.

• Upstream Interference Elimination: Target samples were fully homogenized in an SDS-sonication matrix and reduced with 1,4-dithiothreitol (DTT) to open up the glycoprotein structure. To prevent signal suppression from N-linked species, N-glycans were first exhaustively cleaved using peptide N-glycosidase F (PNGase F). The liberated N-glycans were then separated from the remaining O-glycoproteins via an advanced Hypersep column chromatography system.
• Controlled Chemical Release & Derivatization: The isolated, intact O-glycoprotein fraction was subjected to rigorous reductive β-elimination at 56°C overnight to chemically liberate the O-glycans as stable alditols. Following purification through sequential ion-exchange and C18 solid-phase extraction, residual borates were strictly removed. The purified native O-glycans were then permethylated, a crucial derivatization step that replaces all reactive hydroxyl and amino hydrogens with methyl groups, significantly enhancing ionization efficiency and stabilizing fragile sialylated glycoforms.
• High-Throughput MALDI-TOF-MS Analysis: The permethylated O-glycan samples were crystallized and analyzed on a state-of-the-art MALDI-TOF-MS operating in positive ion mode. This provides exceptional sensitivity, rapid data collection, and clean monoisotopic mass profiles ideal for resolving complex O-glycan structures.

Results

The optimized O-glycan analytical workflow yielded a high-fidelity, comprehensive data package for the client's structural profiling and comparative glycomics:

• High-Resolution MALDI-TOF-MS Chromatogram: Generated clean, high-intensity mass spectra with precise mass-to-charge (m/z) signatures, enabling unambiguous structural profiling of the carbohydrate landscape.

Fig.3 MALDI-TOF-MS of sample O-glycans. (CD BioGlyco)

• Definitive O-Glycan Identification: Individual mass peaks were mapped to specific O-glycan structural compositions (such as Core 1 through Core 4 structures), resolving complex branching configurations.

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

• Comprehensive O-Glycan Feature Distribution: The assay delivered a detailed quantitative summary of major structural traits, classifying the O-glycoform pool based on critical biological features including fucosylation, sialylation, and core type distribution.

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

CD BioGlyco provides a comprehensive and accurate O-glycan profiling as well as many customized services, the most advanced technology and well-trained researchers provide guarantee for your glycobiology research. 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. Li, Z.; Cai, W.G. Mucin O-glycan microarrays. Current Opinion in Structural Biology. 2019, 56: 187-197.