Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.
Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.
Gangliosides are defined as glycosphingolipids bearing one or more sialic acid residues1. They are expressed primarily at the cell surface with their hydrophobic ceramide lipid moiety embedded in the outer leaflet of the plasma membrane and their hydrophilic glycans extending into the extracellular space2. Although distributed widely in vertebrate cells and tissues, they are particularly abundant in the vertebrate brain3, where they were first discovered and named4.
The structures of ganglioside glycans vary and are the basis for their nomenclature (Figure 1). Ganglioside glycans are comprised of a neutral sugar core bearing different numbers and distributions of sialic acids. The smallest ganglioside, GM4, has only two sugars (sialic acid bound to galactose)5. Larger naturally occurring gangliosides may contain well over a dozen total sugars6 or up to seven sialic acids on a single neutral core7. Their ceramide lipid moieties also vary, having different sphingosine lengths and a variety of fatty acid amides. In the vertebrate brain four ganglioside species, GM1, GD1a, GD1b, and GT1b predominate. Ganglioside expression is developmentally regulated, tissue specific, and cell type specific.
Figure 1: Major brain gangliosides and their biosynthetic precursors. Structures are shown using Symbol Nomenclature for Glycans11. Please click here to view a larger version of this figure.
Gangliosides function at the molecular level by engaging and modulating proteins in their own membranes (cis regulation) or by engaging glycan binding proteins in the extracellular milieu, including bacterial toxins and lectins on other cells (trans recognition)3. Specific binding of gangliosides to regulatory proteins and/or self-association with other molecules into lipid rafts results in changes in cell behavior that impact nervous system structure and function, cancer progression, metabolism, inflammation, neuronal proteinopathies, and infectious diseases8. Because of their diverse cellular roles, methods for their isolation and analysis can provide enhanced insights into the regulation of physiological and pathological processes. Here, validated methods for rapid small-scale extraction and analysis, and preparative scale isolation of gangliosides from brain are provided. Opportunities and challenges for application to other tissues are discussed.
Tissue collection was performed under conditions authorized by the Johns Hopkins Animal Care and Use Committee.
1. Small scale ganglioside extraction and partial purification
CAUTION: Use appropriate ventilation when working with volatile and toxic solvents. Avoid plastic throughout; solvents will extract chemical components from many plastics that interfere with subsequent analyses. Polytetrafluoroethylene (PTFE) is an exception; PTFE-lined closures should be used to cap glass storage vials.
2. Large scale ganglioside extraction and purification
CAUTION: When working with volatile solvents, use explosion resistant blenders. Do not use plastics except PTFE. Tetrahydrofuran, chloroform, and ethyl ether are toxic volatile organic compounds. Work in a fume hood with protective gloves and safety goggles.
Time (min) | %A | %B |
0 | 100 | 0 |
7 | 100 | 0 |
12 | 63 | 37 |
82 | 54 | 46 |
82.01 | 0 | 100 |
92 | 0 | 100 |
Table 1: Solvent gradient for HPLC.
3. Thin layer chromatography (TLC) analysis of gangliosides
CAUTION: Chloroform is a toxic volatile organic compound. Work in a fume hood with protective gloves and safety goggles.
Figure 2: Ganglioside TLC equipment and set up. A twin trough chamber is filled to ≈ 0.5 cm on both sides with running solvent. The plate is placed against one side with the origin end immersed in the running buffer. The chamber is covered with an acrylic box to avoid air currents. Panel A, side view prior to plate insertion. The solvent level is visible a few mm above the chamber bottom; Panel B, front view during development. The solvent front is visible at about 40% of the way up the plate. Please click here to view a larger version of this figure.
Figure 3: TLC plate of resolved mixed ganglioside. TLC plate of resolved mixed ganglioside standards (left lane) and purified mixed bovine grey matter gangliosides (right lane) after resorcinol staining and heating with glass cover plate clipped in place. Standard gangliosides (top to bottom) are GM3, GM2, GM1, GD3, GD1a, GD1b, GT1b and GQ1b. After cooling, the plate can be imaged and/or the cover plate taped in place for storage. Please click here to view a larger version of this figure.
The methods described in section 1 (small scale) provide gangliosides at sufficient quantity and purity for qualitative and quantitative determination of major brain gangliosides. Recovery from mouse brain is ~ 1 µmol ganglioside per g brain wet weight (1 nmol/µL) when prepared as described. TLC resolution of 1 µL (1 nmol) using section 3 provides ample material for resorcinol detection and resolves all of the major brain gangliosides as shown for wild type and genetically modified mice in Figure 4. Although mixed gangliosides prepared using section 1 are not free of other major lipids, gangliosides are of sufficient purity for mass spectrometric (MS) determination as shown in Figure 5, either as native purified gangliosides in negative mode or after permethylation in positive mode9. Since section 1 avoids alkaline hydrolysis to remove phospholipids, it retains alkali-sensitive natural modifications, such as O-acetylated sialic acids (see GT1b-OAc, Figure 4)9.
Figure 4: TLC of mouse brain gangliosides. TLC of mouse brain gangliosides from wild-type (WT) and St3gal single and double-null mice purified as in Protocol 1. This figure has been modified from Sturgill et al, 20129. Please click here to view a larger version of this figure.
Figure 5: MS of permethylated wild-type and St3gal2/3-double-null mouse brain gangliosides purified as in section 1. Note that GD1a and GD1b resolve by TLC (Figure 4) but have the same mass so are not distinguishable by one-dimensional MS. This figure has been modified from Sturgill et al, 20129. Please click here to view a larger version of this figure.
Large scale purification (section 2) includes extraction, saponification (to remove phospholipids) and HPLC resolution to provide purified major brain gangliosides GM1, GD1a, GD1b and GT1b suitable for biological experiments and for further chemical and enzymatic modifications. An exemplary HPLC profile and subsequent TLC analysis are shown in Figure 6. Alkali treatment (saponification) is necessary in this protocol to hydrolyze and remove contaminating phospholipids (Figure 7) but will also hydrolyze natural modifications of gangliosides, such as O-acetylated sialic acids, which may be important in some contexts10. For these applications, alternate effective methods for removal of phospholipids from isolated gangliosides have been published6.
Figure 6: Representative HPLC of bovine brain gangliosides. The elution gradient (%B, dotted line) is overlaid on the absorbance (A215, solid line) for the first 75 min of the cycle. Peaks (A215) were collected (numbers in brackets) and subjected to TLC as in Protocol 3. Lane numbers refer to the peak numbers on the chromatogram. Please click here to view a larger version of this figure.
Figure 7: TLC plate of resolved mixed ganglioside. TLC plate of resolved mixed gangliosides standards (lane 1) along with post-saponification partitioned gangliosides (lane 2) and released fatty acids (lane 3). Lipids, including gangliosides, are detected with p-anisaldehyde stain. Standard gangliosides (top to bottom) are GM3, GM2, GM1, GD3, GD1a, GD1b, and GT1b. Please click here to view a larger version of this figure.
The methods for small and large scale ganglioside extraction and isolation reported here are not unique – there are many different solvent extraction and purification approaches that provide excellent results12. The methods reported here for small scale purification from brain, from Fredman and Svennerholm13, were shown to optimize recovery and have proven to be robust and straightforward over many years in our laboratory. Isolation and purification suitable for TLC and MS can be readily completed, from intact tissue to isolated gangliosides, in a few hours. MS can be performed on native purified gangliosides in the negative mode or after permethylation in the positive mode (Figure 7, e.g., see Sturgill et al.9). The yield is very consistent, ≈ 1 µmol ganglioside (≈ 2 µmol ganglioside-bound sialic acid) per g fresh brain tissue for most mammals. The method for large scale extraction and partition from brain reported here, introduced by Tettamani et al.14, is selected to minimize the volume of ganglioside-containing solvent at the first partition (ether-tetrahydrofuran-water). This simplifies subsequent steps that can become cumbersome with techniques that generate large volumes of partitioned gangliosides at the first steps. The HPLC method described, from Gazzotti et al.15, has relatively high capacity and good resolution of the major brain gangliosides.
The small-scale protocols described are well suited for both cells and tissues and are scalable. Since the final steps are reverse-phase capture, evaporation and redissolving in methanol, they can be applied at an arbitrary scale to small samples, such as cultured nerve cells. In this case, we scrape and homogenize cells into 1 mL of water for convenience and proceed with methanol and chloroform addition to generate the appropriate ratios for extraction and then partition. The final dried gangliosides after reverse phase can be redissolved in just a few microliters and analyzed by TLC and MS.
Attention to solvent ratios is critical to success in extraction and solvent partitioning steps for ganglioside isolation. For small scale extraction and partitioning, different chloroform-methanol-water ratios were tested and the ones that generated near quantitative ganglioside isolation are reported here13. Variations from the described ratios will diminish recovery and/or purification. Likewise, attention to solvent ratios in TLC developing solvents is critical. The chloroform-methanol-water ratios reported result in a single clear phase. Small variations may yield cloudy developing solutions which should not be used but can be clarified by dropwise addition of methanol with swirling. Although different extraction and partition solvents are common, solvent ratios should be carefully followed for each variation12. Alterations in TLC solvents are also common to optimize separation of specific gangliosides16. A relatively simple way to modify TLC migration of gangliosides is to change the aqueous phase of the solvent mixture. Using aqueous ammonium hydroxide or calcium chloride instead of potassium chloride alters the ganglioside salt form and relative TLC migration17.
Whereas all vertebrate cells and tissues express gangliosides, the brain and nerve cells are unusual in the high amounts expressed. The protocols described here are applicable to other tissues and cells, but modifications may be required due to the lower abundance and potential contamination with other lipids. These methods as applied to human neutrophils6 and mouse adipose18 provide examples.
The authors have nothing to disclose.
This work was supported by National Institutes of Health (NIH) Common Fund for Glycoscience grant U01CA241953. MJP was supported by the Chemistry-Biology Interface Program at Johns Hopkins (T32GM080189).
Bovine brain, stripped | PelFreez | 57105-1 | |
Ganglioside standards | Matreya | GM1, 1061; GD1a, 1062; GD1b, 1501; GT1b, 1063 | |
Glass bottle with PTFE-lined cap | Fisher Scientific | 02-911-739 | |
Glass centrifuge bottle | Fisher Scientific | 05-586B | |
Glass culture tubes, 16 x 125 mm | VWR | 60825-430 | for collecting HPLC fractions |
Glass separatory funnel (2 L) | Pyrex | 6400-2L | |
Injection syringe – Hamilton 1750 gastight 500 µl | Hamilton | 81265 | |
p-Anisaldehyde, 98% | Sigma-Aldrich | A88107 | |
Potter-Elvhjem Homogenizer | Fisher Scientific | 08-414-14A | Choose appropriate volume option |
Reprosil 100 NH2 10µm 5x4mm guard columns | Analytics-Shop | AAVRS1N-100540-5 | |
Reprospher 100 NH2, 5 μm, 250 mm x 20 mm HPLC column | Analytics-Shop | custom packed | other sizes available |
Resorcinol | Sigma-Aldrich | 30752-1 | |
Rotary evaporator | Buchi | R-300 | |
Sample loop for Model 7725 Injector (5 ml) | Sigma-Aldrich | 57632 | |
Sep-Pak tC18 Cartidges Vac 35 cc (10 g) | Waters | WAT043350 | |
Sep-Pak tC18 Plus Short Cartridge, 400 mg | Waters | WAT036810 | |
Spotting syringe – Hamilton 701N 10 µl | Hamilton | 80300 | |
Thick-walled 13-mm diameter test tubes with PFTE lined caps | Fisher Scientific | 14-933A | |
Threaded 2-ml vials with PFTE lined caps | Fisher Scientific | 14-955-323 | For ganglioside storage |
TLC plates, HPTLC Silica gel 60 F254 Multiformat | Fisher Scientific | M1056350001 | Fluorescence impregnation (F254) stabilizes the sorbent surface |
TLC reagent sprayer | Fisher Scientific | 05-723-26A | |
TLC running chamber for 10 x 10 cm plates | Camag | 22.5155 | |
Waring 1-Liter Stainless Steal Explosion Resistant Blender | Waring | E8520 |