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O-Glycan Linkage Analysis Service

O-linked glycosylation has commonly found in secreted and membrane-bound proteins and takes place in the Golgi. It refers to the binding of glycans to serine and threonine, following with a extent to hydroxyproline and hydroxylysine. The main type of O-glycosylation in secreted and membrane-bound proteins appears to be the addition of reducing terminal N-acetylgalactosamine (GalNAc). The O-linked glycan is also recognized as as 'mucin-type' glycan. In addition to the mucin-type O-linked glycans, many mammalian proteins present mannose (Man), fucose (Fuc), glucose (Glc), Gal or xylose (Xyl) as reducing terminal linkages. This group of O-linked glycan dominated protein localization and transportation, protein solubility, antigenicity and cell-cell interactions.

O-Glycan Linkage Analysis Figure 1. The core structures of mucin-type.

Linkage analysis is a well-established and ingenious approach for linkage positions determination. The principle is to introduce a stable substituent (an ether-linked methyl group) onto each free hydroxyl group of the native glycan. The glycosidic linkages, which are much more labile than the ether-linked methyl groups, are then cleaved by acid hydrolysis, producing individual methylated monosaccharides with free hydroxyl groups at the positions that were previously involved in a linkage. The partially methylated monosaccharides are ring-opened with a reducing agent (normally borodeuteride) to introduce a new hydroxyl group, likely a deuterium atom at C-1, which aids to identify the reducing end of each monosaccharide. All the free hydroxyl groups are then acetylated resulting in partially methylated alditol acetates (PMAAs) that can be identified by GC-MS.

Overview of O-Glycan Linkage Analysis

Creative Proteomics attempts to improve O-glycosylation analysis in four aspects, including O-glycoproteins/peptides enrichment, O-glycan dissociation, O-glycan structure identification and quantitative analysis.

Sample preparation
O-glycans release
Methylation of the released glycans
Glycans are cleaved with exoglycosidases or acid hydrolysis
Reduction and derivatization of free hydroxyl groups
Analyze and quantify by GC-MS

Figure 2. The workflow of our O-glycan linkage analysis.

Samples Delivery

If you are providing a tissue sample, please send the tissue to us with dry ice.
If you are providing a protein sample, use normal tissue or cell lysate for protein extraction.

The types of samples	Protein	Cell	Animal tissue	Blood (EDTA anticoagulation)	Serum	Urine	Microbial samples
Sample size	100 μg	1 x 10⁷ cells	1 g	1 mL	0.2-0.5 mL	2 mL	200 mg (dry weight)

If you have any questions regarding sample delivery, please feel free to contact us and our experts are always available.

Creative Proteomics, with highly experienced bio-scientists and advanced analytical techniques in omics research, can provide our clients with a range of glycomics and bioinformatics analysis services. Please feel free to contact us and see how we can help you address your problems.

* For Research Use Only. Not for use in diagnostic procedures.

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From Our Clients

"I recently used their proteomics service for a project analyzing protein interactions in yeast models. The team was very responsive and helped clarify the methodology they employed, which made me feel confident in the results. The data quality was solid, with clear identification of several key proteins involved in our study. Their thorough analysis enabled me to pinpoint specific interactions that I hadn't considered before, which significantly improved the direction of my research. I appreciate their professionalism and support throughout the process."

Sarah Thompson, University of California, Berkeley

"Our lab collaborated with them on a project studying cancer biomarkers. The proteomics analysis provided was detailed and focused, specifically highlighting the differential expression of proteins between healthy and tumor samples. Their clear explanations of the data helped my team understand the biological implications. I also appreciated their willingness to revise the reports based on our feedback, ensuring that we had everything we needed for our publication. This collaborative spirit was invaluable."

Emily Rodriguez, Stanford University

"Our lab worked with them on a project studying the effects of diet on gut microbiota using proteomics. They used a label-free quantification method to analyze proteins in fecal samples before and after dietary intervention. The results showed significant changes in protein expression linked to microbial activity. This was pivotal for our hypothesis about diet-microbiota interactions. The clarity of their data presentation made it easy for our team to integrate these findings into our ongoing research."

Dr. Lisa Wong, University of Toronto

"My experience with Creative Proteomics during the mass spectrometry analysis was excellent. We sent in human saliva and mouse brain tissue samples, which they expertly analyzed using both LC-MS and GC-MS techniques. The results were invaluable, revealing key metabolites in the saliva and identifying biomarkers linked to brain function in the brain tissue."

Dr. Emily Carter, Senior Research Scientist

"The overall service from Creative Proteomics was outstanding. They made the entire process seamless and efficient, allowing us to focus on our research. We worked with leaf and root samples from various Arabidopsis genotypes for targeted metabolomics analysis. Their thorough profiling of primary and secondary metabolites gave us important insights into how the plants respond metabolically to environmental stress."

Dr. Laura Henderson, Plant Physiologist

"We had a pleasant collaboration with Creative Proteomics on mass spectrometry analysis of lipids. They conducted a detailed analysis of lipid species, providing us with important insights into lipid metabolism and its relationship with metabolic syndrome disease states."

Dr. Sarah Mitchell, Research Scientist

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