Name: mass spectrometric analysis of glycosphingolipid antigens
Uploaded: 2013-04-16T12:00:00
Duration: 13 min 9 s
Description: Watch this Scientific Journal Video about Mass Spectrometric Analysis of Glycosphingolipid Antigens at JoVE.com

DZ is supported by MD Anderson Cancer Center and NIH grants AI079232. MD Anderson Cancer Center is supported in part by NIH grant CA16672.

1. Lab Safety Concerns
<ol>
	<li>All organic solvents must be stored in specific areas with clear labeling. See labels by manufactures for types of fire extinguishers required.</li>
	<li>Several reagents used are potentially carcinogenic, and may cause mental depression. These reagents must be handled in a chemical hood with ventilation (See table of specific reagents and equipment for specifics).</li>
	<li>It is recommended that when working with organic solvents and cells, personal protective equipment such as gloves, lab coat, and eye protection be utilized at all times.</li>
</ol>
2. Extraction of Glycosphingolipids from Rat Leukemia Cells
<ol>
	<li>Store RBL rat leukemia cells8 (10 million to 100 million) in 16x100 mm glass tubes under -80 &deg;C conditions. Cells are counted by a hemocytometer after trypan blue staining. Viability of cells should be higher than 95%.</li>
	<li>Extract the lipids using extensive sonication four times for 1-2 hr each with mixed polarity solvents. Use 6 ml of chloroform-methanol 1:1 (v/v) as the first and last solvent. Six milliliter of isopropanol:hexane:H2O 55:25:20 (v/v/v, remove upper phase by aspiration before use) can then be used as the second and third solvent. This solvent is prepared by mixing isopropanol:hexane:H2O in a 55:25:20 ratio, shaking it vigorously, and allowing an upper phase to appear, which will be removed. Keep the lower phase for use.</li>
	<li>Follow sonication with centrifugation to pellet the insoluble material. Pool the supernatants. Dry them in a Speedvac for 4 hr. Nitrogen stream or rotary evaporator can be used if Speedvac is unavailable. Use a good cold trap to hold evaporated organic solvents. Contamination of organic solvent into vacuum pump oil will degrade the efficiency of the vacuum pump.</li>
	<li>Perform a preliminary analysis using high performance thin layer chromatography (HPTLC). To quantify glycolipids, prepare a 0.2% Orcinol in 50% sulfuric acid solution (100 mg in 50 ml) and a Galactose standard (40 mg in 100 ml stock solution). Prepare in 30 &mu;l reaction volume a series of dilutions to use as standard curve (from 0 g/L to 20 g/L). Add 20 &mu;l of Methanol to each dilution series sample. Dissolve each glycolipid sample in 200 &mu;l of Methanol, sonicate, and use 20 &mu;l for glycolipid quantity measurement. In 1.5 ml Eppendorf tubes with screwed caps (Sarstedt, 72.692.005), add 0.4 ml of 0.2% Orcinol in 50% sulfuric acid solution to each sample, with a series of Galactose-H2O-Methanol solutions which serve for standard curve. Boil for 5 min in heating block at 100 &deg;C. Read optical density of color reaction at 440 nm using Sunrise Tecan Microplate Reader. To perform HPTLC, use chloroform:methanol:H2O 60:35:8 (v/v/v) as the solvent for the neutral lipids treated with chloroform-methanol 1:1 (v/v). Use isopropanol:hexane:H2O 55:25:20 (v/v/v) as the solvent for the acid lipids (gangliosides) . The isolated neutral GSLs was dissolved in 200 &mu;l solvent chloroform:methanol 1:1(v/v) and loaded on Merck silica gel thin layer chromatography plate by Drummond Microcaps. The plates were run in solvent chloroform:methanol:H2O 60:35:8 (v/v/v) and dried. The glycosphingolipids were visualized by Orcinol-sulphuric acid at 300 &deg;C.</li>
	<li>In order to separate out the neutral lipids from the acidic lipids, fraction out the neutral and acidic lipids by anion exchange chromatography on a small column of DEAE Sephadex A-25 [the DEAE Sephadex A25 resin can be prepared by soaking Sephadex A-25 resin powder in chloroform:methanol:H2O 30:60:8 (v/v/v) overnight. Aspirate the supernatant until the Sephadex A25 resin is all that is left. Add fresh chloroform:methanol:H2O 30:60:8 (v/v/v) to wash the resin. Repeat three times to remove the negatively charged residues from Sephadex A25 resin].</li>
	<li>Dissolve, sonicate (for no more than 10 min), and resuspend the lipids from step 2.1 in chloroform:methanol:H2O 30:60:8 (v/v/v) when needed.</li>
	<li>Prepare a small DEAE Sephadex A25 (Cl Form) column by using glass wool and a 9" Pasteur pipette. Pack the glass wool carefully so that resin powder does not pass through the column. Add DEAE Sephadex A25(Cl Form) resin to the "neck" of the 9" Pasteur pipette without letting the column dry. Make sure that you do not see any resin in eluent. The samples dissolved in chloroform:methanol:H2O 30:60:8 (v/v/v) were dissolved. The neutral lipid fraction is the flow through fraction.</li>
	<li>Elute the acidic lipid fraction (bound to the resins) with 8 ml of sodium acetate in methanol. Dry both the neutral and acidic fractions, desalt them by dialysis, dry them by Speedvac and analyze them by HPTLC.</li>
</ol>
The acidic fraction contains gangliosides, which can be dialyzed for the Step 3 reaction directly. The neutral fraction contains not only neutral glycosphingolipids, but also phospholipids (phosphatidylcholine and sphingomyelin), which must be removed by an acetylation reaction.
In our experience, the method of a dialysis cassette, is more efficient and convenient than a dialysis tube or reverse phase C18 column chromatography.
<ol start="9">
	<li>The next step is the removal of phospholipids from neutral glycosphingolipids by an acetylation and peracetylation reaction. Dry the DEAE Sephadex A-25 pass-through fraction in the Speedvac (with cooling) for 4 hr. After the samples are dry, put them back into the Speedvac and dry for another 4 hr. Ensure that these samples are completely dry, as any residual water will prevent the peracetylation reaction.</li>
	<li>Peracetylate the samples with 1 ml of pyridine and 0.5 ml of acetic anhydride in the dark at room temperature or 37 &deg;C overnight. This amount of pyridine and acetic anhydride is proper for up to 200 &mu;g of glycosphingolipids. This reaction can be carried out at 37 &deg;C, as the GSLs have better solubility.</li>
	<li>Dry the peracetylated material by Speedvac for 3 hr, with the addition of 1 ml of toluene 3x (coevaporation) to ensure complete evaporation. If the samples are still not completely dry, Speedvac until dry.</li>
	<li>Prepare a Florisil (Sigma-Aldrich) column (30-60 mesh, 10&times;80 mm) in a Pasteur pipette with glass wool, using Florisil beads. Florisil beads should be completely dried before use, by incubation in a 110 &deg;C oven. Fill the beads into the Pasteur pipette up to the neck, and equilibrate in 1,2-dichloroethane-hexane 4:1 (v/v).</li>
</ol>
Elution from the Florisil column is very fast. Rapid volatility of the solvents can lead to column drying. Thus all solvent mixtures should be prepared before running the column since it is not possible to stop the column during the elution.
<ol start="13">
	<li>Apply the peracetylated sample in 1 ml of 1,2-dichloroethane-hexane 4:1 (v/v), and wash the column with 6 ml of the same solvent, followed by 10 ml of 1,2-dichloroethane. Elute neutral peracetylated GSLs with 6 ml of 1,2-dichloroethane-acetone 1:1 (v/v). This step removes the peracetylated phospholipids from glycosphingolipids, because the peracetylated glycosphingolipids bind to the Florisil column, while peracetylated phospholipids do not.</li>
	<li>Dry the fractions by Speedvac for 2 hr and deacetylate with 2 ml 0.5 M sodium methoxide [Sigma-Aldrich, 403067] in 2 ml of methanol for 3 hr at room temperature.</li>
	<li>Neutralize the mixture with 3 ml of methanolic acetic acid [acetic acid/methanol 15/85 v/v], dry by Speedvac, desalt by dialysis [Dissolve the dried products in 3 ml of water, and inject into the Slide-A-Lyzer Dialysis Cassette, dialyze against water for 24 hr. Change the water at least twice]. Dry the dialyzed GSLs (in water solution) by Speedvac until the samples are completely dry.</li>
</ol>
3. Chemical Modification (Permethylation) of Glycosphingolipids13
<ol>
	<li>Introduce GSLs (1-20 &mu;g) to a glass tube with a screw cap and Teflon liner, and add dimethyl sulfoxide (150 &mu;l) without using special drying conditions or inert gas atmosphere.</li>
	<li>Prepare powdered sodium hydroxide (40-60 mg) from sodium hydroxide particles by grinding them in a chemical mortar with a pestle. Make sure to store the powders in a very dry environment and be careful to do the grinding in the chemical hood to avoid breathing the powders.</li>
	<li>Next add the sodium hydroxide powder to the sample solution with a spatula. Add iodomethane (80 &mu;l) with a 100 microliter Halmilton syringe, [or a 100 microliter VWR calibrated pipette linked to a syringe through rubber tubing can be used], and shake the mixture at room temperature for 1 hr.</li>
	<li>Quench the methylation reaction with water (2 ml). Extract the permethylated products 3 times by the addition of dichloromethane (2 ml) [glycolipids are in the dichloromethane phase, which is the lower phase, so the upper phase can be transferred to a new glass tube, and extracted again with dichloromethane].</li>
	<li>Wash the combined dichloromethane extracts 3 times with 2 ml water [ the upper phase, which is the water, can then be disposed of]. Following the final wash, transfer the samples to a new tube, and dry using the Speedvac.</li>
	<li>Samples can then be dissolved in 100 to 200 &mu;l of methanol for ESI-MS analysis.</li>
</ol>
4. MALDI-TOF Analysis of the Permethylated Gycosphingolipids
<ol>
	<li>Perform MALDI-TOF-MS and MALDI-TOF/TOF-MS/MS experiments on a MALDI TOF-TOF Mass Spectrometer (Applied Biosystems 4700, Foster City, CA). Prepare the matrix by mixing 50% volume of 10 mg/ml dihydroxybenzoic acid (DHB) in acetonitrile and 50% volume of 0.1% trifluoroacetic acid (TFA) in water.</li>
	<li>The DHB matrix can be spotted on the MALDI target (1 &mu;l), followed by a 1 &mu;l (containing 100 ng GSLs) spot of sample dissolved in methanol. Apply slowly and allow it to dry before analysis.</li>
</ol>
5. Ion Trap MS Analysis of Permethylated Glycosphingolipids
<ol>
	<li>Carry out mass spectrometry on a linear ion trap mass spectrometer (LTQ, ThermoScientific, San Jose, CA) using a nanoelectrospray source, 0.30 &mu;l/min flow rate at 230 &deg;C capillary temperature in positive ion mode with 30-50% collision energy. Set collision energies to leave a minimal residual abundance of precursor ion. Detect all ions as sodium adducts.</li>
	<li>Identify the iGb3 (Gal&alpha;3Gal&beta;4Glc&beta;1Cer) in an isobaric mixture of Gb3 (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and iGb3 (Gal&alpha;3Gal&beta;4Glc&beta;1Cer) standards by comparing the different patterns of MS4 product ions from the sodiated molecular ion via the glycan fragment m/z 667 and the terminal disaccharide 1-ene ion m/z 445 (i.e. X&rarr;667&rarr;445&rarr;, X means the molecular ion detected at MS1) of pure permethylated iGb3 (Gal&alpha;3Gal&beta;4Glc&beta;1Cer) standards via ESI-LIT-MS with those of permethylated Gb3 (Gal&alpha;3Gal&beta;4Glc&beta;1Cer).</li>
</ol>

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B., Hakomori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+methylation+analysis+of+glycolipids+using+capillary+gas+chromatography-chemical+ionization+mass+fragmentography+with+selected+ion+monitoring.">Microscale methylation analysis of glycolipids using capillary gas chromatography-chemical ionization mass fragmentography with selected ion monitoring.</a> Methods Enzymol. 138, 13-25 (1987).</li><li>Spooncer, E., Fukuda, M., Klock, J. C., Oates, J. E., Dell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+polyfucosylated+lactosaminoglycan+from+human+granulocytes.">Isolation and characterization of polyfucosylated lactosaminoglycan from human granulocytes.</a> J. Biol. Chem. 259 (8), 4792-4801 (1984).</li><li>Ciucanu, I., Costello, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+oxidative+degradation+during+the+per-O-methylation+of+carbohydrates.">Elimination of oxidative degradation during the per-O-methylation of carbohydrates.</a> J. Am. Chem. Soc. 125, 16213 (2003).</li><li>Platt, F. M., Neises, G. R., Karlsson, G. B., Dwek, R. A., Butters, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-butyldeoxygalactonojirimycin+inhibits+glycolipid+biosynthesis+but+does+not+affect+N-linked+oligosaccharide+processing.">N-butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharide processing.</a> J. Biol. Chem. 269 (43), 27108-27114 (1994).</li><li>Mulloy, B., Hart, G. W., Stanley, P., Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+47+Structural+Analysis+of+Glycans.">Chapter 47 Structural Analysis of Glycans.</a> Essentials of Glycobiology. , (2009).</li><li>Ashline, D., Singh, S., Hanneman, A., Reinhold, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congruent+strategies+for+carbohydrate+sequencing.+1.+Mining+structural+details+by+MSn.">Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MSn.</a> Anal. Chem. 77 (19), 6250-6262 (2005).</li><li>Zhang, H., Singh, S., Reinhold, V. 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DZ is a consultant for BioTex, Houston, TX, and an inventor involved in patents related to technologies mentioned in this article, issued or in application.

The glycome, a term coined in analogy to the genome and proteome, refers to all saccharide structures of an organism. To fully comprehend the manifold functions of glycosylation will require an integrated approach including both functional and structural glycomics studies. Both are complicated by the non-template driven nature of glycan biosynthesis, the resulting complexity and diversity of glycan structures, the frequent involvement of aglycone structure in modulating glycan ligand-receptor interactions at the molecular level, and the functional importance of low abundance ligands.
It is generally accepted that mass spectrometry (MS) is an indispensable method for structural glycomics studies, especially for identifying and characterizing low abundance ligands. To observe glycans or glycoconjugates as molecular species, we often use a highly efficient, low energy ionization method, called electrospray ionization (ESI). For more detailed characterization of glycan structure, it is essential to select individual molecular species and break the glycans into smaller pieces. This is normally done by collision-induced dissociation, which involves activating the glycans through collision with inert gas molecules. The increased energy induces bond breakage, and systematic analysis of the resulting fragments provides information about the molecular structure of the glycan. Often, "signature" fragments can be generated that are diagnostic for particular glycan structural features. Together with the molecular mass, these fragments can sometimes be sufficient for identifying glycans, but in the past much more information has been required to fully characterize them, particularly if the structure is novel. These methods include sugar composition analysis, gas chromatography mass spectrometry analysis of partially methylated alditol acetates for linkage analysis, and specific glycosidase digestion17.
As demonstrated over the past decade18-19, multiple rounds of low energy fragmentation, which can be effectively carried out in an ion-trap mass spectrometer (IT-MS), greatly improves the information yield from glycan mass spectral analysis. With four or five rounds of fragmentation (which cannot be done with other MS instruments), it is possible to distinguish isomeric glycans that contain the same sugar components arranged in different sugar linkages, even when these are present together in mixtures of components with the same molecular mass (isobars). For this analysis, the glycans were derivatized, replacing all free hydroxyl groups with methyl groups using permethylation. While structural assignment of the glycans is possible without permethylation, the permethylation increases sensitivity17.
Levery and Zhou, have combined all of the potential advantages of IT-MS methodology, including the detection of isomeric structures using signature diagnostic ions, observable only in MS4 and MS5 spectra, for the highly sensitive identification and quantitation of glycosphingolipids present in the form of multiple isobaric mixtures7-9. In theory, we may be able to identify every existing glycolipid structure, pending the availability of standard glycolipids, which can be chemically synthesized.
The critical steps of this analysis are the recovery rate of GSLs from biological samples. Typically 80 &mu;g of GSL can be recovered from 100 million tumor cells such as RBL. To generate sufficient molecular ions for multiple rounds of fragmentation in ion-trap mass spectrometers, at least 10 microgram of tumor GSLs are required. Low yield of GSLs during purification will lead to low abundance of ions, which could not be subjected to further fragmentation. The success of analysis is dependent on the amount of GSLs which are recovered. Typically, final concentration of permethylated GSLs should reach 1 mg/ml, dissolved in methanol, before being analyzed on a nano-electrospray source. The limit for a thorough MSn analysis is dependent on the abundance of specific ion in MS1 profile. A typical e7 ion abundance is required for a thorough MS5 analysis. The low yield of GSLs during purification can be caused by loss of GSLs during peracetylation step (which removes the phospholipids), when the per-acetylated GSLs must be bound to Florisil resin chromatography column, washed, and subsequently eluted. To ensure the binding of per-acetylated GSLs to silica gel, samples should be reloaded twice to the chromatography column after flowing through.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>1,2 Dichloroethane</td>
 <td>Sigma-Aldrich</td>
 <td>34872</td>
 <td>Carcinogenic </td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>VMR</td>
 <td>JT9524-0</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic anhydride</td>
 <td>Fluka</td>
 <td>45830</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fischer</td>
 <td>A18P-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform </td>
 <td>Fischer</td>
 <td>C606-1 </td>
 <td>Carcinogenic</td>
 </tr>
 <tr>
 <td>DEAE Sephadex A25 (Cl Form)</td>
 <td>GE Healthcare Biosciences</td>
 <td>AB 17&mdash;170-01 </td>
 <td>100 gram</td>
 </tr>
 <tr>
 <td>Decane (anhydrous): </td>
 <td>Sigma-Aldrich</td>
 <td>457116 </td>
 <td>100 ml</td>
 </tr>
 <tr>
 <td>Dichloroacetic acid</td>
 <td>Sigma</td>
 <td>D54702 </td>
 <td>Carcinogenic</td>
 </tr>
 <tr>
 <td>Dialysis cassette </td>
 <td>Fischer(Pierce) </td>
 <td>66110 </td>
 <td>Slide-A-Lyzer, 3500 MWCO, 3-12 ml </td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Thermo Scientific </td>
 <td>20684 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl acetate </td>
 <td>Fischer</td>
 <td>UN1173 </td>
 <td></td>
 </tr>
 <tr>
 <td>Florisil</td>
 <td>Fluka</td>
 <td>46384 </td>
 <td>for packing chromatography column</td>
 </tr>
 <tr>
 <td>Hexanes </td>
 <td>EMD</td>
 <td>HX0290-6 </td>
 <td></td>
 </tr>
 <tr>
 <td>Iodomethane </td>
 <td>Riedel de Haen, Germany</td>
 <td>03810 </td>
 <td>stabilized with silver foil Carcinogenic</td>
 </tr>
 <tr>
 <td>Methanol </td>
 <td>VWR/EMD</td>
 <td>MXO475-1 </td>
 <td></td>
 </tr>
 <tr>
 <td>Neutral glycosphingolipid Qualmix </td>
 <td>Matreya</td>
 <td>1505</td>
 <td></td>
 </tr>
 <tr>
 <td>Monosialganglioside mixture</td>
 <td>Matreya</td>
 <td>1508</td>
 <td></td>
 </tr>
 <tr>
 <td>Disialoganglioside mixture </td>
 <td>Matreya</td>
 <td>1509</td>
 <td></td>
 </tr>
 <tr>
 <td>Lactosyl ceramide and sialosylderivatives </td>
 <td>Matreya</td>
 <td>1510</td>
 <td></td>
 </tr>
 <tr>
 <td>Gangliotetraosyl ceramide and sialosyl derivatives </td>
 <td>Matreya</td>
 <td>1511</td>
 <td></td>
 </tr>
 <tr>
 <td>Pasteur pipettes</td>
 <td>Fischer</td>
 <td>13-678-6A </td>
 <td>9"''</td>
 </tr>
 <tr>
 <td>Pyridine: </td>
 <td>Sigma</td>
 <td>270407 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Hydroxide: </td>
 <td>BDH </td>
 <td>0292 </td>
 <td>500 gram</td>
 </tr>
 <tr>
 <td>Sodium methoxide </td>
 <td>Sigma </td>
 <td>404367 </td>
 <td>0.5 M solution in methanol</td>
 </tr>
 <tr>
 <td>Toluene </td>
 <td>J.T. Baker</td>
 <td>9351-03 </td>
 <td>4 L</td>
 </tr>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Pasteur pipettes</td>
 <td>Fischer</td>
 <td>13-678-6A </td>
 <td>9"''</td>
 </tr>
 <tr>
 <td>Fiber glass (glass wool)</td>
 <td>Corning Incorporated</td>
 <td>3950 </td>
 <td>Pyrex 9989 glass</td>
 </tr>
 <tr>
 <td>12x75 mm glass tubes</td>
 <td>Fischer</td>
 <td>14-962-10B</td>
 <td></td>
 </tr>
 <tr>
 <td>Calibrated pipettes (100 microlitter)</td>
 <td>VMR</td>
 <td>53432-921</td>
 <td></td>
 </tr>
 <tr>
 <td>16x100 mm disposible borosilicate tubes x1,000 </td>
 <td>Fischer</td>
 <td>14-961-29 </td>
 <td>With screw caps</td>
 </tr>
 <tr>
 <td>Caps for glass tubes</td>
 <td>Kimble</td>
 <td>40566C </td>
 <td>size/13-415 </td>
 </tr>
 <tr>
 <td> Merck TLC plates</td>
 <td>Sigma</td>
 <td>Z 29, 301-6</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass tank for TLC</td>
 <td>Sigma</td>
 <td>Z 12,619-5 </td>
 <td></td>
 </tr>
 <tr>
 <td>Drummond Microcaps </td>
 <td>Drummond scientific company</td>
 <td>1-000-0100 </td>
 <td>10 &mu;l, for loading of TLC samples</td>
 </tr>
 <tr>
 <td>Sunrise Microplate Absorbance Reader</td>
 <td>Tecan</td>
 <td>A-5082</td>
 <td></td>
 </tr>
 <tr>
 <td>Whatman Chromatography Paper</td>
 <td>Fischer</td>
 <td>05-716-3E</td>
 <td>18 cm x 34 cm For TLC Tank</td>
 </tr>
 <tr>
 <td> Orcinol ferric chloride spray reagent</td>
 <td>Sigma</td>
 <td>O7875</td>
 <td>For detecting glycosphingolipids on TLC plates</td>
 </tr>
 <tr>
 <td>Prevel Spray Unit</td>
 <td>Sigma</td>
 <td>Z 36,555-6 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sonicator</td>
 <td>Fischer</td>
 <td>FS20 </td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Sorvall</td>
 <td>Legend RT </td>
 <td></td>
 </tr>
 <tr>
 <td>Speedvac</td>
 <td>Savant</td>
 <td>AS160 </td>
 <td>With chemical trap for organic solvants</td>
 </tr>
 <tr>
 <td>MALDI TOF-TOF Mass spectrometer</td>
 <td>Applied Biosystems</td>
 <td>Proteomics 4700 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ion Trap Mass spectrometer</td>
 <td>Thermo</td>
 <td>LTQ </td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

An example of a MALDI MS analysis of the glycosphingolipids from rat leukemia cell line RBL (an antigen presenting cell type that presents glycosphingolipid antigens to NKT cells) is shown in Figure 3. The ions can be assigned to specific glycosphingolipid structures (Figure 3A). In RBL cells treated by NB-DGJ16, a drug which inhibits glycosphingolipid synthesis, significant reduction of all glycosphingolipids was observed (Figure 3B). Structural isomers cannot be distinguished. The MS/MS capacity of the Applied Biosystems 4700 allows fragmentation of MS1 ions to MS2 fragments, allowing limited structural information to be generated. However, MS2 analysis is often not sufficient to discriminate oligosaccharide isomers.
The Ion-Trap MS analysis of glycosphingolipids enables the study of isomers, such as iGb3 (Gal&alpha;3Gal&beta;4Glc&beta;1Cer) and Gb3 (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) which may exist as the trihexosylceramide ions detected in Figure 3A. To detect such isomers, standard iGb3 and Gb3 were analyzed by multiple rounds of fragmentation, until differences were found (Figure 4A and 4B). In the example presented here (Figure 5C) iGb3 and Gb3 could be discriminated by comparing to standard iGb3 and Gb3.
<img alt="Figure 1" src="/files/ftp_upload/4224/4224fig1.jpg" /> 
Figure 1. Flow chart of the glycosphingolipid extraction procedure, and chemical modification of glycosphingolipids (permethylation). Glycosphingolipids were extracted from cells by organic solvents. To remove non-glycosphingolipids, lipids can be peracetylated and deacetylated. Second, glycosphingolipids were chemically modified by a permethylation reaction, which improves sensitivity and specificity of MS detection. Finally, the glycosphingolipid profiles were determined using MALDI-TOF mass spectrometry and ion trap mass spectrometry.
<img alt="Figure 2" src="/files/ftp_upload/4224/4224fig2.jpg" /> 
Figure 2. Glycosphingolipid Quantification by Orcinol Sulfuric Acid Method: Quantification of glycosphingolipids using a 0.2% Orcinol in 50% sulfuric acid solution and a Glucose Standard Curve.
<img alt="Figure 3" fo:content-width="4.75in" fo:src="/files/ftp_upload/4224/4224fig3highres.jpg" src="/files/ftp_upload/4224/4224fig3.jpg" /> 
Figure 3. Glycosphingolipidomics of rat leukemia cells. A. Representative MALDI mass spectrum; each ion represents a specific glycosphingolipid structure, structural isomers cannot be distinguished. B. NB-DGJ, a drug that inhibits glycosphingolipid synthesis, significantly reduces all glycosphingolipids produced in RBL cells.
<img alt="Figure 4" fo:content-width="4in" fo:src="/files/ftp_upload/4224/4224fig4highres.jpg" src="/files/ftp_upload/4224/4224fig4.jpg" /> 
Figure 4. Tandem Mass Spectrometry of isomers of Glycosphingolipids. A. Characteristic decomposition pathways for positive mode ion trap mass spectrometry of permethylated GSLs Gb3Cer and iGb3Cer. Gb3 and iGb3 are clearly separated by their fragmentation patterns. The differences in linkage are reflected in the MS4 product ions consistent with the isobaric m/z 445 precursors. B. Representative results showing iGB3 can be measured in a mixture of iGb3/Gb3 isomers. Stars indicate diagnostic ions for iGb3 only. <a href="https://www.jove.com/files/ftp_upload/4224/4224fig4large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/4224/4224fig5highres.jpg" src="/files/ftp_upload/4224/4224fig5.jpg" /> 
Figure 5. Ion trap MS allows analysis of isomers of glycosphingolipids. A. Standard iGb3 and Gb3 show difference of fragmentation profile after 4 rounds of fragmentation in an ion trap LTQ mass spectrometer. B. Characteristic MS4 profile of ions derived from iGb3 or Gb3. C. Trihexosyleramides are mixtures of iGb3 and Gb3 isomers which can be identified by ion trap MS method (Figure by Dapeng Zhou). <a href="https://www.jove.com/files/ftp_upload/4224/4224fig5large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about Mass Spectrometric Analysis of Glycosphingolipid Antigens at JoVE.com

Mass Spectrometric Analysis of Glycosphingolipid Antigens

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 ...

mass-spectrometric-analysis-of-glycosphingolipid-antigens

Research

JoVE Journal

Immunology and Infection

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...