Gangliosides chromatography

J. Muthing, High resolution thin-layer chromatography of gangliosides, J. Chromatogr. A., 720(1-2) (1996) 3-25. 

Buffered Tetrahydrofuran. In 1973, Tettamanti et al. [19) described an improved procedure for the extraction, separation and purification of brain gangliosides. In this method, the brain tissue was subjected to homogenization and extraction with buffered [potassium phosphate buffer, pH 6.8) tetrahydrofuran. Following centrifugation, diethyl ether was added and the mixture separated into organic and aqueous phase. The gangliosides, recovered exclusively in the aqueous phase, were then freed of residual phospholipids and other minor contaminants [i.e. peptides)by column chromatography on silica gel.This procedure, as shown by the authors,was superior to the commonly used chloroform/methanol... 

Glycolipid analysis. Gangliosides were extracted with chloroform-methanol (2 1), purified on Sephadex G-25 columns and separated into individual species by thin-layer chromatography as described previously (12). 

Column Chromatography. Sepharose beads containing covalently linked gangliosides (0.2 ml packed volume) were placed into a pasteur pipette containing a small amount of glass wool. Columns were washed with HEM containing 50 ug/ml bovine serum albumin (3 ml). Interferon solutions in MEM-albumin (1 ml) were placed on the columns, which were eluted with MEM-albumin at a flow rate of no more than one drop per minute. Fractions of 1 ml were collected and interferon titers determined in each fraction after serial two-fold dilution. Columns onto which mouse fibroblast interferon had been loaded, were eluted with MEM-albumin first, then with 0.07 M N-acetylneuraminyl lactose at pH 2. 

This problem was approached by incorporating radioactive percur-sors into the glycolipids of both brain and lymphoma cells of the Thy-1.2 and 1.1 types. We have used C-palmitate as a per-cursor of ceramide, and - C-N-acetylmannosamine as a percursor of sialic acid (7). Glycolipids were isolated and the radioactive gangliosides were resolved by two-dimensional thin layer chromatography in two different solvent systems followed by autoradiography. 

Chromatography was done as described in Figure 3, using the gangliosides derived from 2 X 10s cells. Assayed fractions are labeled with numbers that correspond to the anti-Thy-1 PFC assay in Figure 6. Number 10 refers to the area surrounding the spots that were assayed (15,). 

Further characterization of Thy-1 active glycolipids was accomplished by fractionating brain gangliosides into mono-, di- and trisialo-gangliosides by DEAE column chromatography according to the procedure described by Nagai (14). These fractions eluted from the column were tested for Thy-1 activity (Table III). 

V. B. Ivleva, Y. N. Elkin, B. A. Budnik, S. C. Moyer, P. B. O Connor, and C. E. Costello, Coupling thin-layer chromatography with vibrational cooling matrix-assisted laser desorption/ ionization Fourier transform mass spectrometry for the analysis of ganglioside mixtures, Anal Chem., 76 (2004) 6484-6491. 

The compounds represented in this facsimile were as follows Nl, neutral lipids (cholesterol, triacylglycerol) PE, phosphatidylethanolamine PS, phos-phatidylserine PI, phosphatidylinositol PC, phosphatidylcholine Sph, sphingomyelin X, gangliosides, polyphosphoinositides, lysophosphatidic acids. The chromatography was done on silica gel G plates with chloroform-methanol-ester (65 35 31, v/v) as the solvent. 

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