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>

<ol>
	<li>Schnaar, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biology+of+Gangliosides.">The Biology of Gangliosides.</a> Advances in Carbohydrate Chemistry and Biochemistry. 76, 113-148 (2019).</li><li>DeMarco, M. L., Woods, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic-resolution+conformational+analysis+of+the+GM3+ganglioside+in+a+lipid+bilayer+and+its+implications+for+ganglioside-protein+recognition+at+membrane+surfaces.">Atomic-resolution conformational analysis of the GM3 ganglioside in a lipid bilayer and its implications for ganglioside-protein recognition at membrane surfaces.</a> Glycobiology. 19 (4), 344-355 (2009).</li><li>Schnaar, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gangliosides+of+the+vertebrate+nervous+system.">Gangliosides of the vertebrate nervous system.</a> Journal of Molecular Biology. 428, 3325-3336 (2016).</li><li>Klenk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+die+Ganglioside,+eine+neue+Gruppe+von+zuckerhaltigen+Gehirnlipoiden+[About+gangliosides,+a+new+group+of+sugar-containing+brain+lipids].">&#220;ber die Ganglioside, eine neue Gruppe von zuckerhaltigen Gehirnlipoiden [About gangliosides, a new group of sugar-containing brain lipids].</a> Hoppe-Seyler's Zeitschrift f&#252;r Physiologische Chemie. 273, 76-86 (1942).</li><li>Uemura, S., Go, S., Shishido, F., Inokuchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+machinery+of+GM4:+the+excess+amounts+of+GM3/GM4S+synthase+(ST3GAL5)+are+necessary+for+GM4+synthesis+in+mammalian+cells.">Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells.</a> Glycoconjugate Journal. 31 (2), 101-108 (2014).</li><li>Nimrichter, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-selectin+receptors+on+human+leukocytes.">E-selectin receptors on human leukocytes.</a> Blood. 112 (9), 3744-3752 (2008).</li><li>Saito, M., Kitamura, H., Sugiyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+heptasialosyl+c-series+ganglioside+in+embryonic+chicken+brain:+its+structure+and+stage-specific+expression.">A novel heptasialosyl c-series ganglioside in embryonic chicken brain: its structure and stage-specific expression.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1571 (1), 18-26 (2002).</li><li>Todeschini, A. R., Hakomori, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+glycosphingolipids+and+gangliosides+in+control+of+cell+adhesion,+motility,+and+growth,+through+glycosynaptic+microdomains.">Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1780 (3), 421-433 (2008).</li><li>Sturgill, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+the+major+brain+gangliosides+GD1a+and+GT1b.">Biosynthesis of the major brain gangliosides GD1a and GT1b.</a> Glycobiology. 22, 1289-1301 (2012).</li><li>Cavdarli, S., Delannoy, P., Groux-Degroote, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=O-Acetylated+gangliosides+as+targets+for+cancer+immunotherapy.">O-Acetylated gangliosides as targets for cancer immunotherapy.</a> Cells. 9 (3), (2020).</li><li>Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbol+nomenclature+for+graphical+representations+of+glycans.">Symbol nomenclature for graphical representations of glycans.</a> Glycobiology. 25 (12), 1323-1324 (2015).</li><li>Schnaar, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+glycosphingolipids.">Isolation of glycosphingolipids.</a> Methods in Enzymology. 230, 348-370 (1994).</li><li>Svennerholm, L., Fredman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+the+quantitative+isolation+of+brain+gangliosides.">A procedure for the quantitative isolation of brain gangliosides.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 617, 97-109 (1980).</li><li>Tettamanti, G., Bonali, F., Marchesini, S., Zambotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+procedure+for+the+extraction,+purification+and+fractionation+of+brain+gangliosides.">A new procedure for the extraction, purification and fractionation of brain gangliosides.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 296, 160-170 (1973).</li><li>Gazzotti, G., Sonnino, S., Ghidoni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal-phase+high-performance+liquid+chromatographic+separation+of+non-derivatized+ganglioside+mixtures.">Normal-phase high-performance liquid chromatographic separation of non-derivatized ganglioside mixtures.</a> Journal of Chromatography. 348, 371-378 (1985).</li><li>Schnaar, R. L., Needham, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-layer+chromatography+of+glycosphingolipids.">Thin-layer chromatography of glycosphingolipids.</a> Methods in Enzymology. 230, 371-389 (1994).</li><li>Ledeen, R. W., Yu, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gangliosides:+structure,+isolation,+and+analysis.">Gangliosides: structure, isolation, and analysis.</a> Methods in Enzymology. 83, 139-191 (1982).</li><li>Lopez, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+lacking+sialyltransferase+ST3Gal-II+develop+late-onset+obesity+and+insulin+resistance.">Mice lacking sialyltransferase ST3Gal-II develop late-onset obesity and insulin resistance.</a> Glycobiology. 27 (2), 129-139 (2017).</li></ol>

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. Gangliosiden komen tot expressie op alle gewervelde celoppervlakken en zijn vooral overvloedig aanwezig op zenuwcellen. Ze reguleren celsignalering en celcelherkenning. Dit protocol introduceert ganglioside isolatie en analyse.De technieken werden geselecteerd om de gangliosideopbrengsten op grote en kleine schaal te maximaliseren en tegelijkertijd de zuivering te stroomlijnen. We beschrijven ook methoden om relatief snel kwalitatieve en semikwantitatieve ganglioside analyses uit te vo...

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

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

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 ...

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

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

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

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease ...

Medicine

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

Ganglioside Extraction, Purification and Profiling

Samenvatting

Meer video's verkennen