Ganglioside Extraction, Purification and Profiling

Summary

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.

Abstract

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.

Introduction

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 nomenclatu....

Protocol

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.

1. Extraction
   1. Weigh a single fresh or thawed mouse brain (or sagittal half-brain, ~ 0.2-0.5 g) and pl....

Results

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

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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