Separation Of Gangliosides Research Articles

Enhanced thin-layer chromatographic separation of GM1b-type gangliosides by automated multiple development.

Enhancement in separation of gangliosides on silica gel precoated high-performance TLC plates has been obtained by automated multiple development chromatography. A less polar mixture of the standard solvent chloroform-methanol-20 mM aqueous CaCl2 (120:85:20, v/v) was used. Lowering the water content achieved separation of two complex monosialoganglioside fractions, isolated from murine YAC 1 T lymphoma and MDAY-D2 lymphoreticular cells. Three-fold chromatography in the solvent chloroform-methanol-20 mM aqueous CaCl2 (120:85:14, v/v) resulted in TLC separation of GM1b-type gangliosides, substituted with C24 and C16 fatty acids and with Neu5Ac and Neu5Gc as well, which could not be achieved by unidirectional standard chromatography. Compared to conventional single chromatography, the technique described allows high-resolution separation of extremely heterogenous ganglioside mixtures and offers a convenient tool for both analytical and preparative TLC.

Quantification of gangliosides by microbore high performance liquid chromatography.

A highly sensitive analytical method was developed that allows the separation of ganglioside mixtures and quantification of individual non-derivatized gangliosides in the concentration range between 2 pmol and 1 nmol. Gangliosides were separated with a gradient of acetonitrile/phosphate buffer on a 1 mm diameter microbore HPLC column packed with Spherisorb-NH2. They eluted according to their number of sialic acid residues with increasing phosphate and decreasing acetonitrile concentrations. The separation of different gangliosides with equal sialic acid content is also described. The column effluent was monitored at the maximum of absorption at 197 nm. The sensitivity is higher than resorcinol staining of fractionated gangliosides by thin layer chromatography, previously the standard method for ganglioside analysis. The separated gangliosides can be analyzed by further methods. The HPLC method described here has been applied to the analysis of serum and oligodendroglioma specimens.

S19.12 Improved separation of gangliosides on high performance thin-layer chromatography plates by automated multiple development

S19.12 Improved separation of gangliosides on high performance thin-layer chromatography plates by automated multiple development

High-performance liquid chromatographic separation of gangliosides, combined with immunological detection and fast atom bombardment mass spectrometric characterization

A complete strategy for the isolation of individual mono- and disialogangliosides has been elaborated. We have used straight-phase silica gel chromatography or partitioning to obtain a crude ganglioside fraction. This fraction was then peracetylated and run through a second silica gel column. After anion-exchange chromatography the gangliosides were separated by straight-phase high-performance liquid chromatography with chloroform-methanol-water mixtures as eluting solvents. The method is suitable for preparative isolation of gangliosides and subsequent structural characterization by thin-layer chromatography-enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry and/or gas chromatography-mass spectrometry, which is demonstrated by several examples, including the separation of GalNAc-II3NeuAc-GgOse4Cer from GalNAc-isoII3NeuAc-GgOse4Cer.

Detection of femtomolar quantities of the ganglioside GM1 on thin-layer chromatography plates by native choleratoxin and labeled antisera

After separation of gangliosides by thin-layer chromatography, femtomolar quantities of GM1 were detected by incubating the plate with native choleratoxin, followed by anticholeratoxin and species-specific labeled antiserum. Only stable reagents were involved when antiserum labeled with horseradish peroxidase was used. Native choleratoxin rather than iodinelabeled toxin ensured good reproducibility of the method.

Rapid ion-exchange separation of human brain gangliosides

An extract of human brain gangliosides was separated on a Spheron 1000-DEAE spherous fine-grain macroporous glycolmethacrylate anion-exchanger. Linear gradient elution using ammonium acetate in methanol resulted in the complete separation of mono, di-, tri-, tetra- and pentasialogangliosides in 35 min at the load of 2.4 mg/ml of ion-exchanger. The ganglioside fractions thus obtained were characterized by thin-layer chromatography on silica gel. In conclusion, the scope of rapid separation of gangliosides is discussed, based on a combination of column chromatographic methods.

Autoradiography of ganglioside antigens separated by high-performance thin-layer chromatography with their antibodies.

An improved method is described for the detection of ganglioside antigens with the antibodies on thin-layer chromatograms. The method involves the following steps: Separation of gangliosides on a high-performance thin-layer chromatographic plate, application of antibody solution, and detection of bound antibody with [125I]staphylococcal protein A. Using specific antibodies against gangliosides GM4, GM1, GD3, and asialo GM1 ganglioside, the method allowed positive identification of these antigens on thin-layer plates. It also provides a convenient means of assessing the specificity of an anti-glycolipid antibody.

Separation of gangliosides on a new type of anion-exchange resin

A new anion exchange resin, Spherosil-DEAE-Dextran, consisting of porous glass beads covered with cross-linked DEAE-Dextran, was used in the separation of gangliosides. The gangliosides were eluted from the resin with a discontinuous gradient of potassium acetate in methanol, separating the gangliosides quantitatively into mono-, di, tri-, tetra- and pentasialoganglioside fractions. The new resin was found to have higher binding capacity, to show less unspecific adsorption and to give a better separation of the higher oligosialogangliosides than did DEA-Spherosil, QAE-Sephadex, DEAE-Sephadex and DEAE-Sepharose. Anion exchange chromatography improved discrimination between closely allied gangliosides as well as quantification and identification of individual gangliosides, especially the minor ones. The new procedure was used in the separation of the gangliosides in human infant forebrain and cerebellum.

Isolation and separation of gangliosides on a new form of glass bead ion exchanger.

A new anion exchange resin, Spherosil-DEAE-Dextran, built on porous glass beads covered with crosslinked DEAE-Dextran, has been compared with other resins, DEAE-Sephacel, DEAE-Sephadex and DEAE-Sepharose, regarding its value in the isolation and separation of gangliosides from a crude ganglioside extract. In comparison with the best commercial resin, DEAE-Sepharose, Spherosil-DEAE-Dextran had higher binding capacity, higher binding specificity and gave a better separation of gangliosides with four and five sialic acids. The constant bed volume facilitated regeneration.

Separation of gangliosides, corticosteroids and water-soluble non-lipids from lipid extracts by sephadex columns

Two column chromatographic systems (benzene-Sephadex and hexane-Sephadex) are described which effectively separate water-soluble intermediates from lipid extracts of tissues incubated with radioactive precursors. Biphasic solvent systems were prepared by equilibrating either benzene or hexane with aqueous ethanol. Sephadex G-25 was allowed to swell in the aqueous ethanol (stationary phase). From crude lipid extracts the mobile phase (benzene or hexane) eluted neutral and phospholipids; polar lipids and non-lipids were retained, but were eluted by aqueous ethanol. The hexane-Sephadex column was more effective in separating gangliosides from glycerolipids and separated corticosteroids from sterols, ketosteroids and progestins.

The separation of gangliosides by glass fiber chromatography

The separation of gangliosides by glass fiber chromatography