The overall goal of this procedure is to create a specific and sensitive method to gain insight into the expression profile of glyco finger lipid antigens in immune organs and cells. This is accomplished by first extracting the glyco fing lipids from the cells. The second step is to separate neutral glyco fingering lipids from acidic glyco fingal lipids.
After the phospholipids are removed from the neutral lipids, the extracted glyco lipids are chemically modified using a perm methylation reaction where all hydroxyl groups of hexoses are replaced by methoxy groups. Ultimately, the extracted glyco finger lipids are analyzed using matrix assisted laser, DESORPTION ionization, time of flight, mass spectrometry, abbreviated mold TOF MS, and iron trap mass spectrometry. The main advantage of this technique over existing methods like analytical HPLC and antibody staining, is that mass spectrometry is more sensitive and measures accurate massive molecules.
This method can help answer key questions in the immunology field, such as measuring low abundance of glycolipid antigens. The implications of this technique extend towards therapy or diagnosis of cancer because many glycolipid antigens such as GD two and global H are expressed as cancer biomarkers. I'll be demonstrating the procedure as an undergraduate student from professor DA pen Zoe's laboratory.
Dr.David Hawk will be performing procedures on the mass spectometry instruments Before starting this procedure. Prepare rat basophil leukemia cells counting the cells by a hemo cytometer. After tripper blue staining, the viability of cells should be higher than 95%Extract the lipids by first adding six milliliters of a one-to-one volumetric ratio of chloroform methanol to the cells.
Use extensive sonication for one to two hours with the mixed polarity solvent. Repeat this procedure twice with a 55 to 25 to 20 volumetric ratio of isopropanol hexane water prepared as described in the written procedure as a final extraction. Add six milliliters of chloroform, methanol, and sonicate before.
Follow sonication with centrifugation to pellet the insoluble material at 4, 000 RPM for five minutes. Pull the Senna before drying them in a speed vac for four hours. In order to separate out the neutral lipids from the acidic lipids, use anion exchange chromatography on a small column of de A e sedex A 20.
After preparing the DEAE SEDEX A 25 as described in the written procedure accompanying this video, prepare the column using glass wall and a nine inch pasta pipette. Pack the glass wall carefully so that resin powder does not pass through the column. Add DEAE sedex A 25 resin to the neck of the nine inch past air pipette without letting the column dry.
Make sure there is no resin in the eent. Dissolve sonicate and re suspend the lipids in a 30 to 60 to eight volumetric ratio of chloroform methanol water and apply the dissolved samples to the column. The neutral lipids will flow through the column while the charged lipids will remain in the column elute the acidic lipid fraction with eight milliliters of sodium acetate in methanol.
Shown here is the dried negatively charged fraction after being treated with sodium acetate. Desalt the fractions by dialysis using the slide Eliza dialysis cassette following dialysis. Dry the fractions by speed back before preliminary analysis of the lipids using high performance thin layer chromatography or H-P-T-L-C to perform H-P-T-L-C dissolve the isolated neutral glyco finger lipids or gsl in 200 microliters of a one-to-one volumetric ratio of chloroform methanol solvent.
Load the sample on a silica gel thin layer chromatography plate. Run the plates in a 60 to 35 to eight volumetric ratio of chloroform methanol water after drying the plate. Visualize the glyco finger lipids with arsenal sulfuric acid at 300 degrees Celsius for the acidic lipids or gangliosides.
Use a 55 to 25 to 20 volumetric ratio of isopropanol hexane water as the solvent and run in the H-P-T-L-C in the same manner. At this point, the neutral fraction contains not only neutral glyco finger lipids, but also phospholipids, which must be removed by acetylation and para acetylation reaction. First, dry the DEAE sedex A 25 pass through fraction in the speed VAC with cooling for four hours.
After the samples are dry, put them back into the speed back and dry for another four hours. Ensure that these samples are completely dry as any residue water will prevent the para acetylation reaction. Add one milliliter of PERINE and 0.5 milliliters of acetic an hydride to the samples for the para acetylation reaction of up to 200 micrograms of glyco fing lipids incubating the dark at 37 degrees Celsius overnight.
A temperature at which the GSL have better add one milliliter of toluene to ensure complet evaporation and dry the para ated material by speed vac for three hours. If the samples are still not completely dry, speed VAC until dry shown here is the dried paracetic sample. Meanwhile, prepare a Floris seal column by first completely drying floris seal beads by incubation in a 110 degrees Celsius oven.
Then prepare the column in a pasta pipette with glass wool by filling the beads into the pasta pipette up to the neck and equilibrating in a four to one volumetric ratio of one two di chloro Ethan Hexane Elution from flues cell 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's not possible to stop the column during the elution. Apply the percet sample in one milliliter to a four to one volumetric ratio of one two di chloro ETH Ethan Hexane. Wash the column with six milliliters of the same solvent, followed by 10 milliliters of one two di chloro ethane elute.
The neutral percet GSL with six milliliters of a one-to-one volumetric ratio of one two dichlorine acetone. This step removes the percet phospholipids from glyco fingering lipids because the percet glyco fingering lipids bind into the fluorol column while percet phospholipids do not. After drying the fractions by speed back for two hours, deacetylate with two milliliters of 0.5 molar sodium, meth oxide, and two milliliters of methanol for three hours at room temperature.
Following incubation, neutralize the mixture with three milliliters of methanol, acetic acid and dry by speed back, diesel by dialysis, by dissolving the dried products in three milliliters of water and injecting into the slide Eliza dialysis cassette followed by dialysis against water for 24 hours, changing the water at least twice dry the dialyzed gsl by speed vac until the samples are completely dry For perm methylation. First introduce GSL to a glass tube with a screw top and Teflon liner at 150 microliters of dimethyl sulfoxide without using special drying conditions or inert gas atmosphere in the chemical hood, prepare powdered sodium hydroxide from sodium hydroxide particles by grinding them in a chemical mortars with a pestle. Next, add the sodium hydroxide powder to the sample solution with a spatula.
Then add 80 microliters of IO methane with a 100 microliter Hamilton syringe. Shake the mixture at room temperature for one hour after quenching the methylation reaction with two milliliters of water extract the perm methylated products by the addition of two milliliters of dichloromethane. Glycolipids are in the dichloromethane phase, which is the lower phase, so the upper phase can be transferred to a new glass tube.
The perm methylated products can then be extracted again by chloro methane for a total of three extractions. Wash the combined di chloro methane extracts three times with two milliliters of water, dumping the upper aqueous phase after each wash following the final wash, transfer the samples to a new tube and dry. Using the speed vac samples can then be dissolved in 100 to 200 microliters of methanol for mass spectrometry analysis performed as described in the written procedure samples were submitted to the University of Texas MD Anderson Cancer Center proteomics facility and analyzed by MALDI to MS MS mass spectrometer as well as an ion trap LTQ mass spectrometer.
Shown here is an example of the MALDI MS analysis of the glyco finger lipids from the rat leukemia cell line RBL, an antigen presenting cell type that presents glyco finger lipid antigens to NKT cells. The ions can be assigned to specific glyco fingering lipid structures in RBL cells treated with the drug that inhibits glyco finger lipid synthesis. Significant reduction of all glyco finger lipids was observed.
Structural isomers cannot be distinguished. The msms capacity of the applied biosystems 4, 700 allows fragmentation of MS one ions to MS two fragments allowing limited structural information to be generated. However, MS two analysis is often not sufficient to discriminate oligosaccharide isomers, whereas the ion trap MS analysis of glyco finger lipids enables the study of oligosaccharide isomers to detect such isomers.
Standard IGB three and GB three were analyzed by multiple rounds of fragmentation until differences were found as shown in this characteristic. MS four profile of ions derived from IGB three or GB three when different ratios of IGB three and GB three are mixed, A standard curve can be made based on the ion abundance of signature ions derived from each isomer. In the example presented here, IGB three and GB three could be discriminated by comparing to the standards.
This spectrum represents the MS four analysis of 1 3 2 5, the molecular iron representing IMRs of GB three and IGB three signature ions for IGB three and signature ions for GB three were found suggesting the presence of a mixture of the two isomers. The ratio of IGB three to GB three was calculated by comparison to a standard curve. While attempting this procedure, it's important to remember to fully recover glycolipids during chromatography before pro methylation of the glycolipids.
Make sure at least 80 micrograms of glycolipids are recovered during purification. After watching this video, you should have a good understanding of how to analyze glycolipids by a semi-quantitative mass spectometry method. You'll master the key steps of glycolipid extraction, separation into neutral and acidic fractions, modification by perm methylation reaction, and submitting modified glycolipids to your mass spectometry facility.
Don't forget that working with one two di chloro ethane chloroform, chlor acetic acid, and I oto methane can be extremely hazardous in carcinogenic and precautions such as gloves and lab coat should always be taken while performing this procedure. Respect the rules and enjoy your discovery.
