CD BioGlyco's services have helped clients from all over the world, the efficient Lipidomics Analysis Services and Sphingolipid Analysis Services have gained a good reputation worldwide for our company.
Gangliosides, sialic acid-containing glycosphingolipids, are expressed abundantly in the vertebrate nervous system, especially in neurons. GD belongs to gangliosides that contain several types. GD1 gangliosides, at times considered a reserve pool for monosialoganglioside1 lipids, belong to the major gangliosides of the mammal brain. GD2 is a sialic acid-containing glycosphingolipid expressed primarily on the cell surface. The function of this carbohydrate antigen is not completely understood; however, it is thought to play an important role in the attachment of tumor cells to extracellular matrix proteins. GD3 is the discovered earliest cancer-associated ganglioside that promotes adhesion and invasion of cancers.
Fig.1 GD2: a novel therapeutic target in triple-negative breast cancer. (Shao, et al., 2022)
CD BioGlyco provides chromatographic and mass spectrometric methods to analyze GD. Taking the high-performance liquid chromatography (HPLC) method to evaluate the level of gangliosides in serum as an example, our analysis steps include isolation of blood plasma, lipid extraction and isolation of gangliosides, enzymatic digestion of gangliosides, glycan purification, fluorescent labeling of glycans, HPLC qualitative analysis, and statistical analysis. Based on these steps, flow cytometry analysis is performed. This technique is used to detect fluorescently labeled oligosaccharides derived from serum glycosphingolipids by enzymatic digestion with ceramide glycanase.
Plasma is isolated by centrifugation in a density gradient. After separation, the plasma is collected as the upper phase.
Extract plasma samples or cell pellets with a chloroform/methanol mixture and shake vigorously in glass tubes. The solution is sonicated for several minutes in an ultrasonic water bath, then incubate on ice. After centrifugation, the supernatant is transferred to a clean tube and dried under nitrogen flow. Then, dissolve it in chloroform. Specific lipid fractions are eluted sequentially from the column using chloroform, a methanol/acetone mixture, neutral glycolipids, and methanol (gangliosides). The last fraction, containing gangliosides, is dried in a vacuum concentrator and then dissolve in the buffer.
After the sample is completely dissolved, add the ceramide glycanase solution to react under optimal conditions.
After digestion is complete, glycans in the reaction mixture are separated from ceramides, enzymes, and other contaminants by column purification. The eluate is dried in a vacuum concentrator.
Using 2-amino benzamide to label purified glycan samples.
Dilute the labeled glycan samples by adding HPLC-grade water. Chromatography is performed according to an appropriate HPLC run program.
Qualitative analysis is based on the comparison of the retention times of the test components with the retention times of standards. Based on quantitative analysis of samples containing standards of known GD content, the relationship between fluorescent signal and ganglioside content is established.
To further confirm the quantitative HPLC analysis, flow cytometry is used as a reference method, as it allows quantitative assessment of antigen expression.
At CD BioGlyco, our one-stop Ganglioside Analysis Service has been greatly recognized internationally, and we will continue to explore to meet the analysis needs of clients in the field. We provide high-quality GD analysis using HPLC and MS. If you are interested in our services, please contact us for more details without any hesitation.
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