The overall goal of this procedure is to isolate and identify the triglyceride contents present in bat sebaceous secretions. First, extract the surface lipid fraction from integumentary tissues under conditions to minimize contamination and oxidation. Separate the lipids by thin layer chromatography to obtain the triglyceride fraction.
Now process the spectral data with instrument software and analyze with online chemical databases. Ultimately, preparative thin layer chromatography and matrix assisted laser desorption ionization time of flight mass spectrometry are used to determine differences in triglyceride composition profiles among experimental and controlled groups. This method can be used to help identify secreted lipids that may function as chemical signals in host pathogen interactions.
While we're using this method to investigate the clinical signs of white nose syndrome, this method can be applied to other mammalian systems, including looking at sexual signaling or possibly interspecific differences in milk composition. Clean all instruments with methanol before and between tissue collection from different individuals. Trim about one gram of hair from the skin and place in a 125 milliliter erlenmeyer flask.
To sample the sebum from the wing surfaces. Wet cotton balls with chloroform methanol solvent. Scrub the skin and place these in a separate flask.
Now extract the tissue with 10 milliliters of chloroform methanol containing 0.5%BHT to prevent oxidation after two hours at about 0.5 grams of anhydro sodium sulfate and pass through filter paper to collect the solvent. Repeat the extraction sequentially with one to one and one to two chloroform methanol. Then pool the filtrates, evaporate the pooled filtrates under a stream of nitrogen.
Determine the dry weight and dissolve the lipid residue to a concentration of 10 milligrams per milliliter. Store the sample in glass vials at minus 20 degrees Celsius for up to one month to remove plasticizers that can interfere with later mass spectrometry analysis. Fill 1.5 milliliter micro centrifuge tubes with three to two chloroform methanol.
Then rinse with acetone and air dry store sample tubes in a dust-free container and handle these tubes only with gloves. To prevent contamination from skin oils. Activate the TLC plate by first pre developing it with three to two chloroform methanol.
Add solvent to the TLC chamber to a depth of one centimeter. Then place the plate in the chamber and allow the solvent to run completely to the top of the plate. Remove the plate and dry it in a fume hood until the solvent evaporates.
Then heat the plate at least 10 minutes at 120 degrees Celsius. Now mark the plate with a pencil mark at the top. Place a straight pencil line 1.5 centimeters from the bottom edge of the plate to mark the baseline where the sample and standards will be placed.
Next, cut a piece of filter paper large enough to line the two short walls and one long wall of the TLC chamber. Insert the filter paper liner into the chamber. Pour 100 milliliters of freshly prepared mobile phase solvent into the chamber to a depth of about one centimeter.
Apply a silicone grease seal along the top edge of the chamber. Cover with the glass lid and equilibrate overnight. Apply the sample manually or with an automated sample applicator making a continuous streak.
Use the outer lanes of the TLC plate to spot about 20 microliters mixture of standards. Place the loaded TLC plate into the equilibrated chamber. Close the lid and develop the plate until the solvent runs to the top edge.
After approximately 45 minutes, remove the plate and allow the excess solvent to evaporate in a fume hood Spray with 0.05%rodine six G in 95%ethanol. Then visualize the lipid bands under a long wavelength ultraviolet lamp with a pencil. Mark the RF position for the fluorescent bands resolved in the sample and standards.
Take a photographic record. Next, identify the band corresponding with the tag standard. Transfer it to a large piece of glass and whey paper, and then into a 1.5 milliliter solvent washed micro centrifuge tube.
Add one milliliter of three to two chloroform methanol solvent to the sample and sonicate for one minute. Pellet the silica by centrifugation and then transfer the solvent into a new pre weighed tube. Repeat the one milliliter extraction, pull the filtrates and then evaporate the solvent under a stream of nitrogen.
Store the dried residue containing tags under nitrogen in the dark at four degrees Celsius while additional tissue samples are separated by TLC Rho Domine. Six G is present in samples, but it is insoluble in hexane and is removed during sample dissolution.Immediately. Before MS analysis, prepare the 500 fmol per microliter A CTH working solution and 10 milligram per milliliter tag standards as described in the text protocol.
Also, prepare the 0.5 molar stock solution of DHB with 90%methanol for use as sample and standard matrix. Cover the tube with aluminum foil to protect it from light. Now combine 10 microliters of DHB matrix, 10 microliters of sample or standard, and five microliters of one molar sodium hydroxide mix, and briefly centrifuge the tube to bring the mixture to the bottom spot.
One microliter of standard sample or A CTH onto a MALDI stainless steel target plate and place in a desiccate to dry. Then place the target plate into the instrument for data acquisition. Perform the MALDI toff MS analysis in positive reflection mode.
Tune and calibrate the instrument as described by the instrument's manufacturer using the A CTH and tag standard solutions. Acquire spectra for each sample spotted on the target plate after smoothing and subtracting the background from Aldi Spectra process ion peaks manually with the online search engine lipid maps, copy and paste the list spectra into the list of precursor ions and intensity box. Limit the search to AAL composition desired Identify tags by the mass charge ratios from ions present in the spectrum for each sample.
If fatty acid methyl ester percentages are available from a separate GCMS analysis, add these data to obtain probabilities of tags. Present mammalian integumentary lipid analysis by preparative TLC with HEA as the mobile phase usually resolves into four distinct bands corresponding to sters FFAs tags and sterile esters wax EERs squalene. These multi to mass spectra were obtained for tags isolated from the eastern red bat.
Typical for a preparation from non aquatic mammals. The spectra contain ion peaks in the mass range between master charge ratio eight 50 to nine 10. Fatty acid moieties are the dominant AAL constituents and tags.
Further information on AAL ratios can be deduced by observing the peaks in the D AAL glycerides region of the spectrum, which represent tag fragmentation. The MALDI spectrum displays three distinct tag regions where master charge ratio is based on number of carbons. Eastern red bat wing tissue is characterized by dominant peaks in these three regions at master charge ratio 8 53 0.7 8, 79 0.7, and 907.8.
Remember that when you're working with live or euthanized bats, you must obtain all required permits for the handling, storage, and transportation of these animals. Also, you must obtain permission from your institutional animal care and use committee as well as the institutional biosafety committee. If live bats or nervous tissue from bats is being handled, remember that all handlers should be vaccinated for rabies.
Also, when working with certain bath bat pathogens such as geo MiSiS ants, a biosafety level two lab is required.
