Since O-Glycosylation occurs in all domains of life, the enzymatic release of O-glycosylated protein is a key segment in glycomics research. CD BioGlyco integrates various advanced technologies to develop an efficient and accurate release strategy of O-glycosylated proteins. Welcome clients from all over the world to cooperate with us!

O-Glycosylated Protein

Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently linked to amino acid side chains. The most common types of glycoproteins are N-linked and O-linked glycoproteins, which differ by structural differences. Glycans are attached to specific amino acid residue side chains of proteins in a co-translational or post-translational manner, a process known as glycosylation. These post-translationally linked glycans have multiple roles, such as cell recognition, adhesion, signaling, and more. O-Glycosylation is a post-translational event in which the carbohydrate is covalently attached to the hydroxyl group of serine (Ser) or threonine (Thr). O-Glycosylation provides ligands for selectins, resists proteolysis, and is involved in recognition phenomena. O-Glycoproteins change their glycosylation patterns when expressed in new cellular systems resulting in impaired protein function. In addition, O-glycosylation has implications for the biotechnology industry. Therefore, it is of interest to know the location and type of O-glycosylation in a given glycoprotein.

Various forms of O-glycosylation are abundant in organisms and play important roles in many cellular functions. Changes in O-glycosylation are also closely associated with a variety of diseases, such as Alzheimer's disease, diabetes, and cancer. O-Glycosylation changing of Mucin 1 (MUC1) is one of the striking features of oncogenic mucins. Studies have shown that the cancer-associated glycoform of MUC1 contributes to the progression, invasion, and metastasis of epithelial tumors. In conclusion, understanding these O-glycosylation changes in cancer cells may lead to new diagnostic and therapeutic opportunities.

Key Technologies

• High-throughput sample preparation: Process many samples simultaneously, efficiently, and with minimal manual intervention.
• Solid-phase extraction (SPE): Following enzymatic release, the sample contains a complex mixture of the now-free glycans, the deglycosylated protein, and the enzyme itself. SPE is a purification technique used to isolate the glycans of interest from this mixture.
• Size-exclusion chromatography (SEC): Used as a final cleanup step to ensure that the sample is free from any high-molecular-weight contaminants that could interfere with subsequent analyses.

Enzymatic Release of O-Glycosylated Protein: Advanced Solutions for Glycoproteomics

To better understand these O-glycosylation changes in cancer cells, a reliable method must be identified to separate O-glycosylated proteins from native glycoproteins before analysis. At CD BioGlyco, we have developed an advanced Enzymatic Release platform. We provide clients with efficient and accurate release services of native mucin-type O-glycosylated proteins via O-protease. We high-specifically hydrolyze peptide bonds in proteins at the N-terminus of O-glycans at Ser or Thr to release intact native core 1 O-glycosylated proteins. In addition, we provide clients with fast and sensitive O-glycosylated protein characterization services.

Fig.1 The process of enzymatic release of O-glycosylated protein. (CD BioGlyco)

• Sample Preparation

Upon receipt, samples undergo thorough quality assessment to determine protein concentration, purity, and initial glycosylation status.

• Enzymatic Digestion

A customized enzyme cocktail is selected based on the target O-glycan type and protein properties. The reaction is then performed at optimized temperature, pH, and incubation time to ensure efficient and complete release of O-glycosylated proteins.

• Glycan and Protein Separation

After enzymatic digestion, the released O-glycans are separated from the deglycosylated protein and residual byproducts using techniques such as solid phase extraction (SPE) or filtration to obtain highly purified released glycosylated fractions.

• Glycan Derivatization

To enhance detection signals by mass spectrometry (MS) or chromatography, glycans are labeled with 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP), or permethylation to improve ionization efficiency and chromatographic resolution, resulting in more sensitive and accurate analytical results.

• Downstream Glycan Analysis

Structural elucidation and quantification are performed using high-resolution mass spectrometry (MS), while separation and relative quantification are performed using high-performance liquid chromatography (HPLC).

Workflow

Publication Data

Applications

• Identifying novel O-glycosylation-based biomarkers for early disease detection, progression monitoring, and therapeutic response prediction in conditions such as cancer, inflammatory diseases, and neurological disorders.
• Characterizing O-glycosylation patterns of therapeutic proteins (e.g., antibodies, fusion proteins) to ensure product quality, efficacy, and safety.
• Investigating the role of O-glycosylation in pathological processes, including host-pathogen interactions, immune evasion, and cellular signaling pathways, provides insights into disease pathogenesis.

Advantages

• Our proprietary enzyme panels and optimized protocols ensure highly specific and efficient release of O-glycans, minimizing non-specific cleavage and maximizing glycan yield.
• We prioritize methods that maintain the native structure of released glycans, preventing desialylation, degradation, or rearrangement.
• Beyond enzymatic release, CD BioGlyco offers a full suite of downstream glycan analysis services, including advanced MS and chromatography.

Frequently Asked Questions

CD BioGlyco is your trusted partner in glycobiology research, offering a comprehensive suite of services from the enzymatic release of O-glycosylated proteins to advanced glycan analysis and complementary solutions. Please feel free to contact us to discuss your specific project needs and provide tailored solutions.

Associated Services