Name: ganglioside extraction, purification and profiling
Uploaded: 2021-03-12T15:00:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Ganglioside Extraction, Purification and Profiling at JoVE.com

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).

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.
<ol>
	<li>Extraction
	<ol>
		<li>Weigh a single fresh or thawed mouse brain (or sagittal half-brain, ~ 0.2-0.5 g) and place in a Potter-Elvehjem homogenizer prechilled in a bucket of ice. 
		NOTE: Previously frozen brains can be used after thawing at 0-4 &#176;C.</li>
		<li>Add 4.1 mL per g tissue wet weight of water and homogenize with 10 strokes. 
		NOTE: Accurate solvent ratios are key to optimal extraction and partition, the goal is chloroform-methanol-aqueous (4:8:3) assuming brain tissue is 80% aqueous.</li>
		<li>Add 13 mL per g tissue wet weight of methanol, shift to ambient temperature (22 &#176;C) and mix. 
		&#8203;NOTE: The solution will appear cloudy. Addition of methanol at this step, without chloroform, optimizes protein precipitation. All subsequent steps are at ambient temperature (22 &#176;C).</li>
		<li>Transfer to a thick-walled glass screw-capped tube with a PTFE-lined screw cap at ambient temperature (22 &#176;C) and mix thoroughly. Add 6.5 mL per g tissue wet weight of chloroform, cap, and mix thoroughly. Centrifuge at 450 x g for 15 min. Transfer the clear supernatant to a fresh screw-capped tube and measure the volume, &#34;recovered extract volume&#34;.</li>
	</ol>
	</li>
	<li>Partition
	<ol>
		<li>Add 0.173x &#34;recovered extract volume&#34; of water to the clear supernatant, cap, vortex vigorously, and centrifuge as described in step 1.1.4. 
		NOTE: The goal is to have chloroform-methanol-aqueous in the ratio 4:8:5.6. The mixture will be cloudy and resolve into two phases: an upper aqueous-rich phase and a lower chloroform-rich phase at ~ 4:1 ratio. Wait for 60 min or centrifuge at 450 x g for 15 min for complete phase separation.</li>
		<li>Transfer the upper phase, which contains the gangliosides, into a fresh glass tube with a PTFE-lined screw cap.</li>
	</ol>
	</li>
	<li>Reverse phase cartridge chromatography
	<ol>
		<li>Using a 5 mL glass syringe, wash a tC18 solid phase extraction cartridge (400 mg) with 3 mL of methanol, then 3 mL of chloroform-methanol-water (2:43:55). Load the upper phase from step 1.2.2 onto the tC18 cartridge using&#160;the same glass syringe, collect the flow-through and reload it onto the column to optimize adsorption.</li>
		<li>Using the glass syringe, wash the cartridge with 3 mL of chloroform-methanol-water (2:43:55) then 3 mL of methanol-water (1:1).</li>
		<li>Elute the gangliosides with 3 mL of methanol into a fresh screw-capped tube. Evaporate to dryness under a gentle stream of nitrogen at &#8804; 45 &#176;C. Dissolve in methanol at 1 mL per g of original tissue wet weight.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Extraction
	<ol>
		<li>Thaw frozen bovine brain at 4 &#176;C for several hours. Dissect the grey matter from meninges and white matter. 
		NOTE: The following procedure is described for 100 &#177; 20 g of isolated brain grey matter and is scalable.</li>
		<li>Place 100 g of brain grey matter in a blender and add 1 mL per g brain wet weight of chilled 10 mM potassium phosphate buffer pH 6.8. Homogenize on low for 20 s. Add 8 mL tetrahydrofuran per g brain wet weight and homogenize on low for 10 s. Decant into glass centrifuge bottles and centrifuge at 5,000 x g for 15 min at ambient temperature (22 &#176;C).</li>
		<li>Collect the supernatant, measure its volume, and transfer to a glass separatory funnel. Add 0.3 mL of ethyl ether per mL of the supernatant. Shake vigorously, then allow to sit undisturbed for 30 min during which two phases, an upper ether phase and a lower aqueous phase, separate. Collect the lower phase, which contains the gangliosides, into a glass bottle with a PTFE-lined cap.</li>
		<li>To the upper (ether) phase remaining in the separatory funnel, add 0.1 mL water per mL of original supernatant (step 2.1.3). Shake vigorously, allow phases to separate, collect the lower (aqueous) phase and combine with the previous lower phase. Evaporate the combined lower phases to a dry powder and weigh.</li>
	</ol>
	</li>
	<li>Saponification
	<ol>
		<li>Add 10 mL of 100 mM aqueous NaOH per g powder in a sealed tube. Mix and incubate at 37 &#176;C for 3 h. Allow to cool and adjust to pH 4.5 by dropwise addition of 100 mM aqueous HCl. Measure the volume and transfer to a glass separatory funnel. 
		NOTE: Sialic acids are acid labile; avoid acidification below pH 4.5.</li>
		<li>Based on the aqueous volume, add 2.67 volumes of methanol, mix gently, then add 1.33 volumes of chloroform to create a single-phase solution of chloroform-methanol-aqueous (4:8:3). Mix well.</li>
		<li>Based on the original aqueous volume, add 2.6 volumes of water to bring the mixture to chloroform-methanol-aqueous to a ratio of 4:8:5.6. Shake vigorously, then allow to sit undisturbed to separate two phases, a polar upper phase containing the gangliosides and a nonpolar lower phase. Collect the upper phase in a glass bottle with a PTFE-lined cap. 
		NOTE: Non-sialylated lipids will not appear on thin layer chromatography (TLC) plates stained using resorcinol but will appear when using a p-anisaldehyde stain. The purpose of this saponification is to remove the O-acetylated compounds such as phospholipids.</li>
	</ol>
	</li>
	<li>Reverse phase chromatography
	<ol>
		<li>Pre-wash a large scale (10 g) tC18 solid phase extraction cartridge by passing 50 mL of each of the following three solvents through the column using vacuum or pressure (&#60;1 min each wash): methanol, methanol-water (1:1), then chloroform-methanol-water (2:43:55). Load the upper phase from step 2.2.3 onto the column by vacuum or pressure, collect the flow through, reload, and collect the flow through.</li>
		<li>Wash the column with 30 mL of chloroform-methanol-water (2:43:55), then 30 mL methanol-water (1:1), then elute the gangliosides with 50 mL methanol, and again with 10 mL methanol, collecting each wash and each elution separately. Using TLC (below) confirm that ganglioside is absent from the flow through and washes and eluted in the first elution (50 mL methanol). Evaporate the eluted gangliosides to a dry powder and weigh. 
		NOTE: The purpose of tC18 chromatography is to separate gangliosides from both less and more polar contaminants. Mixed brain ganglioside yield after saponification is ~ 120 mg per g dry brain extract (step 2.1.4). Appearance of gangliosides by TLC in the flow through or washes indicates the solid phase extraction column was saturated. After methanol elution, the column may be further eluted with chloroform-methanol (1:1) to capture less polar lipids.</li>
	</ol>
	</li>
	<li>HPLC purification of individual gangliosides
	<ol>
		<li>Prepare HPLC Solvent A: acetonitrile-5 mM aqueous sodium phosphate buffer pH 5.6 (83:17) and Solvent B: acetonitrile-20 mM sodium phosphate buffer, pH 5.6 (1:1). Degas both solvents for 5 min.</li>
		<li>Pre-equilibrate an HPLC column (20 x 250 mm column packed with amine bonded (NH2) silica spheres, 5 &#181;m diameter, 100 &#197; pore size) with 100% Solvent A for 20 min at 5 mL/min. Set a UV HPLC column effluent detector to 215 nm.</li>
		<li>Dissolve the ganglioside powder from the reverse phase eluate in water at 5 mg/mL. Inject 0.5 mL of the ganglioside mixture onto the HPLC and run the solvent gradient (Table 1) at 5 mL/min, collecting fractions. Gangliosides will appear as A215 peaks with retention times (major brain gangliosides) of 25-70 min: GM1 &#8776; 28 min; GD1a &#8776; 38 min; GD1b &#8776; 46 min; GT1b &#8776; 65 min. Re-equilibrate 20 min with Solvent A after each run. Analyze fractions by thin-layer chromatography.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time (min)</td>
			<td>%A</td>
			<td>%B</td>
		</tr>
		<tr>
			<td>0</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>7</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>12</td>
			<td>63</td>
			<td>37</td>
		</tr>
		<tr>
			<td>82</td>
			<td>54</td>
			<td>46</td>
		</tr>
		<tr>
			<td>82.01</td>
			<td>0</td>
			<td>100</td>
		</tr>
		<tr>
			<td>92</td>
			<td>0</td>
			<td>100</td>
		</tr>
	</tbody>
</table>
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.
<ol>
	<li>Running solvent and TLC plate preparation
	<ol>
		<li>Prepare a running solvent of chloroform-methanol-aqueous 0.25% KCl (60:35:8 by volume). Pour into a 10 cm x 10 cm glass TLC chamber with a stainless-steel cover so that the solvent depth is ~ 0.5 cm. Cover and allow to equilibrate in an area free of air currents for &#62;10 min. 
		NOTE:To isolate the TLC chamber from air currents, an acrylic 5-sided box can be constructed or purchased&#160; (Figure 2). Do not use solvent-saturated filter paper inside the chamber.</li>
		<li>Place a 10 cm x 10 cm or 5 cm x 10 cm silica gel coated glass-backed high performance TLC plate in a drying oven at 125 &#176;C for 10 min. Allow to cool. Use a dulled #2 pencil to draw 5-mm spotting lines with 2-mm separations along a line 1 cm above bottom of the plate and at least 1 cm from either side (Figure 3). Avoid disturbing the silica layer while marking.</li>
		<li>Prepare a standard mix of pure gangliosides in methanol containing 100 &#181;M GM1, 50 &#181;M each of GD1a and GD1b and 33 &#181;M of GT1b. 
		NOTE: This mixture contains 100 pmol of ganglioside sialic acid per &#181;L for each of the four gangliosides, a quantity that provides a strong colorimetric signal by resorcinol staining, which is sialic acid dependent.</li>
	</ol>
	</li>
	<li>Ganglioside resolution
	<ol>
		<li>Wash a 10-&#181;L Hamilton syringe with a beveled needle with methanol. Draw 1 &#181;L methanol into a glass syringe to fill the needle dead volume and then 1 &#181;L of sample or standard. Spot the sample evenly onto the 5-mm premarked lines until &#60;1 &#181;L of solvent (methanol) remains in the syringe. Allow the plate to dry at ambient temperature (22 &#176;C) after all samples are spotted. 
		NOTE: Wash the syringe with methanol between sample loading. An unheated air blower set at low can be used to accelerate drying.</li>
		<li>Place the spotted and dried plate into the preequilibrated TLC chamber with the bottom edge immersed in the running solvent and cover and protect from air currents (Figure 2). Allow the running solvent to advance up the plate by capillary action until the solvent front reaches within 1 cm of the top of the plate. Remove and mark the solvent front at the edge of the plate with a pencil. Allow the solvents to evaporate completely either undisturbed or under mild air flow.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62385/62385fig02.jpg" /> 
Figure 2: Ganglioside TLC equipment and set up. A twin trough chamber is filled to &#8776; 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. <a href="https://www.jove.com/files/ftp_upload/62385/62385fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Ganglioside staining 
	CAUTION: Reagent stains are toxic. Hydrochloric acid is corrosive and toxic. Prepare and spray reagents in a fume hood with protective gloves and safety goggles. 
	NOTE: The plate can be imaged for qualitative image analysis or stored by removing the clamps and securing the cover plate in place with clear tape. Quantitative analysis can be performed by measuring densitometry of ganglioside standards spotted in adjacent lanes.
	<ol>
		<li>Prepare resorcinol spray reagent for the detection of gangliosides based on their sialic acids. Dissolve 6 g of resorcinol in 100 mL water for a 6% resorcinol stock. Dissolve 1 g of CuSO4 in 100 mL of water to make a 1% stock. To 64.7 mL of water add 5 mL of the 6% resorcinol stock, 0.31 mL of the 1% CuSO4 stock then slowly add 30 mL of concentrated HCl and stir gently. May be stored at 4 &#176;C for a month.</li>
		<li>In a chemical fume hood, place the TLC plate with resolved gangliosides, origin end up, in a cut-away cardboard box to protect the walls of the hood from acid spray. Place resorcinol spray reagent in a glass TLC sprayer, attach to a source of pressurized nitrogen, and lightly spray the plate diagonally in the vertical and horizontal directions. Spray the TLC sorbent surface uniformly, but lightly.</li>
		<li>Immediately cover the plate with a clean dry glass cover plate of the same dimensions and secure the cover plate in place with binder clips (Figure 3). Heat the covered plate at 125 &#176;C for 20 min. Gangliosides will appear dark purple against a white background. 
		NOTE: The plate should not appear wet when spraying is complete. Cover plates can be fashioned by scraping the sorbent from previously used TLC plates using a single-edge razor blade. 
		CAUTION: Silica powder is toxic to lungs. Use a mask and dispose silica in a sealed container.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62385/62385fig03.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/62385/62385fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>General lipid staining for gangliosides and phospholipids. 
	&#8203;CAUTION: Sulfuric acid is toxic and corrosive. Addition of concentrated acid to ethanol is exothermic and must be done slowly. Prepare staining reagent in a fume hood with protective gloves and safety goggles.
	<ol>
		<li>Prepare p-anisaldehyde stain by slowly adding 15 mL of concentrated sulfuric acid to 500 mL of ethanol. Stir for 30 min to allow the solution to cool before proceeding. Add 15 mL p-anisaldehyde and stir gently. This may be stored at room temperature (22 &#176;C) up to six months.</li>
		<li>In a chemical fume hood, dip the TLC plate with resolved gangliosides, origin end&#160;down, into a beaker containing the p-anisaldehyde stain. Submerge to the running front for &#8805; 2 s. Remove TLC from stain and allow to drain. Heat the TLC plate on a hot plate at low temperature to develop. 
		NOTE: Lipids will appear dark against a purple background. Staining solution may be recovered for repeated use.</li>
	</ol>
	</li>
</ol>

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The authors claim no competing interests.

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 ...

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62385/62385fig01.jpg" /> 
Figure 1: Major brain gangliosides and their biosynthetic precursors. Structures are shown using Symbol Nomenclature for Glycans11. <a href="https://www.jove.com/files/ftp_upload/62385/62385fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.

<table><tbody><tr><td>Bovine brain, stripped</td><td>PelFreez</td><td>57105-1</td><td></td></tr><tr><td>Ganglioside standards</td><td>Matreya</td><td>GM1, 1061; GD1a, 1062; GD1b, 1501; GT1b, 1063</td><td></td></tr><tr><td>Glass bottle with PTFE-lined cap</td><td>Fisher Scientific</td><td>02-911-739</td><td></td></tr><tr><td>Glass centrifuge bottle</td><td>Fisher Scientific</td><td>05-586B</td><td></td></tr><tr><td>Glass culture tubes, 16 x 125 mm</td><td>VWR</td><td>60825-430</td><td>for collecting HPLC fractions</td></tr><tr><td>Glass separatory funnel (2 L)</td><td>Pyrex</td><td>6400-2L</td><td></td></tr><tr><td>Injection syringe - Hamilton 1750 gastight 500 &amp;micro;l</td><td>Hamilton</td><td>81265</td><td></td></tr><tr><td>p-Anisaldehyde, 98%</td><td>Sigma-Aldrich</td><td>&amp;nbsp;A88107</td><td></td></tr><tr><td>Potter-Elvhjem Homogenizer</td><td>Fisher Scientific</td><td>08-414-14A</td><td>Choose appropriate volume option</td></tr><tr><td>Reprosil 100 NH2 10&amp;micro;m 5x4mm guard columns</td><td>Analytics-Shop</td><td>AAVRS1N-100540-5</td><td></td></tr><tr><td>Reprospher 100 NH2, 5 &amp;mu;m, 250 mm x 20 mm HPLC column</td><td>Analytics-Shop</td><td>custom packed</td><td>other sizes available</td></tr><tr><td>Resorcinol</td><td>Sigma-Aldrich</td><td>30752-1</td><td></td></tr><tr><td>Rotary evaporator</td><td>Buchi</td><td>R-300</td><td></td></tr><tr><td>Sample loop for Model 7725 Injector (5 ml)</td><td>Sigma-Aldrich</td><td>57632</td><td></td></tr><tr><td>Sep-Pak tC18 Cartidges Vac 35 cc (10 g)</td><td>Waters</td><td>WAT043350</td><td></td></tr><tr><td>Sep-Pak tC18 Plus Short Cartridge, 400 mg</td><td>Waters</td><td>WAT036810</td><td></td></tr><tr><td>Spotting syringe - Hamilton 701N 10 &amp;micro;l</td><td>Hamilton</td><td>80300</td><td></td></tr><tr><td>Thick-walled 13-mm diameter test tubes with PFTE lined caps</td><td>Fisher Scientific</td><td>14-933A</td><td></td></tr><tr><td>Threaded 2-ml vials with PFTE lined caps</td><td>Fisher Scientific</td><td>14-955-323</td><td>For ganglioside storage</td></tr><tr><td>TLC plates, HPTLC Silica gel 60 F254 Multiformat</td><td>Fisher Scientific</td><td>M1056350001</td><td>Fluorescence impregnation (F254) stabilizes the sorbent surface</td></tr><tr><td>TLC reagent sprayer</td><td>Fisher Scientific</td><td>05-723-26A</td><td></td></tr><tr><td>TLC running chamber for 10 x 10 cm plates</td><td>Camag</td><td>22.5155</td><td></td></tr><tr><td>Waring 1-Liter Stainless Steal Explosion Resistant Blender</td><td>Waring</td><td>E8520</td><td></td></tr></tbody></table>

ganglioside extraction, purification and profiling

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.

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 &#181;mol ganglioside per g brain wet weight (1 nmol/&#181;L) when prepared as described. TLC resolution of 1 &#181;L (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 <strong class="x...

Watch this Scientific Journal Video about Ganglioside Extraction, Purification and Profiling at JoVE.com

Ganglioside Extraction, Purification and Profiling

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 ...

ganglioside-extraction-purification-and-profiling

Research

JoVE Journal

Biochemistry

4.4K Views.  Johns Hopkins University School of Medicine. 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.

Video: Ganglioside Extraction, Purification and Profiling

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Chemistry

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Neuroscience

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

Ganglioside Extraction, Purification and Profiling

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Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry