Glycan Biosynthesis and Metabolism Analysis

Overview

The main glycosylation types of proteins in mammals can be divided into two types: N-glycosylation and O-glycosylation, most glycoproteins contain only one type of glycosylation. However, some protein peptides have both N-glycan linkage and O-glycan linkage.

• N-glycosylation

The N-glycosylation is covalently linking with the free -NH₂ group of aspartic acid of the protein and the synthesis of N-linked glycans starts with the endoplasmic reticulum (ER), then completes in the Golgi apparatus.

• O-glycosylation

The O-glycan linkage is covalently linked to the free -OH group of serine or threonine of the protein. There has no conservative sequence or fixed core structure on the O-glycosylation site. The O-glycan linkage can be composed of either a monosaccharide or a huge sulfonated polysaccharide, so the analysis of O-glycosylation is more complicated than that of N-glycosylation.

Advantage

• High coverage of glycans
• High sensitivity and few detection restrictions
• High precision
• Advanced platforms and strategies

Service workflow

• Sample collection
• Metabolites extraction
• LC-MS data analysis and bioinformatics analysis
• Generate a report

Fig 2. Workflow of glycan metabolism service of Creative Proteomics.

List of metabolism services of glycans

Sample Requirements

• Serum/plasma: 500 μl/sample

Repeated freezing and thawing of samples must be avoided. The serum sample should be precipitated in the collection tube for 30 minutes at room temperature, then transported to the centrifuge tube and centrifuged at 8000 rpm for 5 minutes. After centrifugation, the supernatant was equally divided into a freezing tube of 500 uL / sample.

• Protein: 100 µg
• Anticoagulated blood (EDTA): 1 mL

Anticoagulants and preservatives must be added immediately after collection and then frozen at -80 °C.

• Urine: 1 ml/sample

Urine samples should be equally divided into centrifuge tubes with 1 mL per tube, each tube is added with 1/100 (w/v) sodium azide and stored at -80 °C.

• Animal tissues: 200 mg/sample

Sample should be frozen in liquid nitrogen immediately and then transported to -80 °C for storage after collected.

• Cells: ≥ 1 × 10⁷/sample

Cytoactive should be terminated immediately to maintaining cell integrity.

• Feces: 500 mg/sample

In general, to assure enough sample to fulfill the whole project, the volume of the single sample need to be offered as much as possible. The remaining samples will be stored for one year free of charge and returned at any time if necessary. All samples need to be stored and transported at-80°C.

Clinical samples are repeated in no less than 30 cases in a single group.

Animal samples are repeated in no less than 9 cases in a single group.

Report Delivery

• Experimental procedure
• Parameters of HPLC and MS
• Raw data, chromatograms and mass spectra
• Metabolites quantification data
• Custom analysis report

Service cycle

• Sample testing: 5-10 working days
• Data analysis: 5-10 working days

Creative Proteomics metabolism analysis platform is committed to the all-around, reliable and accurate analysis service for a variety of target substances, which is suitable for life science research, drug exploration, biological determination and other fields. We provide personalized metabolism analysis service to support your science research.

References

1. Mereiter, S., Balmana, M., Gomes, J., Magalhaes, A., & Reis, C. A. (2016). Glycomic Approaches for the Discovery of Targets in Gastrointestinal Cancer. Front Oncol, 6, 55. doi:10.3389/fonc.2016.00055.
2. Varki, A. (2017). Biological roles of glycans. Glycobiology, 27(1), 3-49. doi:10.1093/glycob/cww086.