Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry

CAUTION: Obtain in advance all requisite state and federal permits for handling, transporting, and storing bats. Approvals must also be obtained from your institutional animal care and use committee, as well as from your institutional biosafety committee. If live bats (or nervous system tissue) are to be handled, animal handlers should be vaccinated for rabies. Bats used in the current study were collected from the Ozark St. Francis National Forest, AR, during summer 2010 according to standard methods (Arkansas State University's Institutional Biosafety Committee approval # 135349-1)²⁸.

1. Tissue Treatment and Lipid Extraction

1. Clean all instruments with methanol before and between tissue collection from different individuals. Trim hair (about 1.0 g) from skin with a scissors and place in a 125 ml Erlenmeyer flask. Sample sebum from wing surface by scrubbing skin with 4 – 6 cotton balls wetted with chloroform:methanol solvent (C:M; 3:2 v/v), and place these in a separate flask.
2. Extract tissue with 10 ml of C:M (2:1 v/v) containing 0.5% butylated hydroxytoluene (BHT) to prevent oxidation²⁹. Only use HPLC quality solvents.
3. After 2 hr, add about 0.5 g anhydrous sulfate to each flask, mix briefly, and collect the solvent by filtering through filter paper.
4. Repeat steps 1.2 and 1.3 twice. Once with 1:1 C:M and sequentially with 1:2 C:M. Pool filtrates together.
5. Evaporate the pooled filtrates under a stream of N₂, determine dry weight, and dissolve the lipid residue in 3:2 C:M (with 0.5% BHT) to a concentration of 10 mg/ml. Store sample in glass vials at -20 °C. It is generally best to analyze samples within one month after sample collection and to minimize freeze-thaw cycles.

2. Lipid Separation by Preparative Thin-layer Chromatography

1. Prepare in advance solvent-washed 1.5 ml microcentrifuge tubes by filling with 3:2 C:M, rinsing with acetone, and air-drying. This is done to remove plasticizers that can interfere with later mass spectrometry analysis. Store sample tubes in a dust-free container and handle these tubes only with gloves to prevent contamination from skin oils.
2. Activate the TLC plate by first pre-developing it with 3:2 C:M. Add enough solvent to the TLC chamber to a depth of 1 cm, then place plate in the chamber (close with glass lid) and allow the solvent to run completely to the top of the plate. This takes about 45 min.
3. Remove the plate and dry in a fume hood until solvent evaporates (about 15 min), then place in an oven for at least 10 min at 120 °C. Place a pencil mark at the top of the plate to maintain orientation when samples are applied. Place a straight pencil line, 1.5 cm from the bottom edge of the plate, to mark the baseline where the sample and standards will be placed.
4. Prepare TLC chamber by cutting a piece of filter paper large enough to line the two short walls and one long wall. Place the filter paper liner into the chamber. It will fully wet when solvent is added to the chamber.
5. Prepare 100 ml of the mobile phase solvent, which is hexane:diethyl ether:acetic acid (H:E:A; 80:20:2 v/v/v). Pour solvent into the chamber to give a depth of about 1 cm. Cover with the glass lid, using a silicone grease seal along the top edge of the chamber. Allow the chamber to equilibrate overnight before use.
6. Apply the sample manually to the prepared plate with a capillary tube or pipette as a continuous streak from about 1.5 cm from one edge to 1.5 cm to the other edge. An automated sample applicator is preferred since it will load the sample in a more homogenous streak. Use the outer lanes of TLC plate to spot about 20 μl mixture of sterol, FFAs, TAGs, and sterol ester standards (use at 10 mg/ml; premade mixtures can be obtained).
7. Place the loaded TLC plate into the equilibrated chamber, close with the lid, and develop the plate until the solvent runs to the top edge. This takes about 45 min with the mobile phase described here. Remove the plate from the chamber and allow the excess solvent to evaporate from the plate in a fume hood for about 1 min.
8. Spray with 0.05% rhodamine 6G in 95% ethanol. Visualize lipid bands under a long wavelength ultraviolet lamp. Mark with a pencil the Rf position for the fluorescent bands resolved in the sample and standards. A photographic record can be taken at this point.
9. Identify the band corresponding with the TAG standard and remove it from the plate by scraping the silica off with a spatula onto a large piece of glassine weigh paper. Transfer silica to a 1.5 ml solvent-washed microcentrifuge tube.
10. Add 1.0 ml of 3:2 C:M solvent to the sample tube, sonicate for 1 min, pellet the silica by centrifugation, and then transfer the solvent into a new pre-weighed tube. Repeat the previous step, pool the filtrates, and evaporate the solvent under a stream of N₂.
11. Store the dried residue containing TAGs under N₂ in the dark at 4 °C while additional tissue samples are separated by TLC. Rhodamine 6G is present in samples, but it is insoluble in hexane and is removed during sample dissolution immediately before MS analysis.

3. TAG Analysis by MALDI-TOF MS

1. Prepare a fresh α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution by dissolving 10 mg CHCA in 1 ml solvent (49.5% ethanol, 49.5% acetonitrile, and 1% aqueous 0.1% TFA).
2. Prepare adrenocarticotropic hormone (ACTH; 18-39 clip, 2465.1989 Da) for instrument resolution and sensitivity testing by mixing 1 μl of ACTH (1 mg/ml) with 39.5 μl of 0.1% trifluoroacetic acid (TFA) to obtain a 10 pmol/μl stock solution. This can be stored at -20 °C for future use.
3. Prepare a fresh working ACTH solution (1 pmol/μl) by taking 1 μl of the 10 pmol/μl stock solution, mixing with 9 μl 0.1% TFA, and then mixing (1:1) with 10 μl of the CHCA matrix solution to obtain a 500 fmol/μl concentration.
4. Prepare TAG standards at 10 mg/ml in 3:2 C:M for MALDI instrument calibration (e.g. tricaprin [470.361 Da], tricaprylin [554.455 Da], trilaurin [638.549 Da], tripalmitin [722.642 Da], tripalmitolein [800.689 Da], trimyristin [806.736 Da], triolein [884.783 Da], tri-11-eicosenoin [968.877 Da], and trierucin [1052.971 Da]).
5. Prepare triolein at 10 mg/ml in 3:2 C:M for an external lock-mass calibration TAG standard.
6. Dissolve the stored TAG samples in hexane to a 10 mg/ml solution.
7. Prepare a 0.5 M (77.06 mg/1.0 ml) stock solution of 2,5-dihydroxybenzoic acid (DHB) with 90% methanol for use as sample and standard matrix. Also prepare a 1.0 M (2.0 g/50.0 ml) solution of NaOH. Cover the tube with DHB solution using aluminum foil to protect from light.
8. Mix (in pre-washed tubes) 10.0 μl DHB matrix, 10.0 μl sample or standard, and 5.0 μl 1.0 M NaOH. Mix and briefly centrifuge the tube to bring mixture to the bottom.
9. Spot 1.0 μl of standard, sample, or ACTH onto a MALDI stainless-steel target plate and place in a desiccator until dry. Place the target plate into the instrument for data acquisition.
10. Perform MALDI-TOF MS analyses in positive reflectron mode. Tune and calibrate the instrument as described by the instrument's manufacturer using the ACTH and TAG standard solutions.
11. Acquire spectra for each sample spotted on the target plate (consistent with instrument specifications; parameters for this work were 5 Hz laser firing rate, ~100 shots per spot to obtain an average spectrum). After smoothing and subtracting the background from MALDI spectra, process ion peaks manually with the online search engine LIPID MAPS (www.lipidmaps.org/tools/ms/glycerolipids_batch).
12. Copy and paste the list spectra into the list of precursor ions and intensity box. Limit the search to acyl composition desired. Identify TAGs by the mass/charge (m/z) ratios from ions present in the spectrum for each sample. If fatty acid methyl ester (FAME) percentages are available from separate GC/MS analysis, add these data to obtain probabilities of TAGs present.

INSTRUMENT: The mass spectrometer used in this study is a Waters MALDI Micro MX (equipped with a 337 nm 20 Hz N₂ laser). Any manufacturer's MALDI instrument with positive reflectron mode capability can be used. General settings used are pulse voltage, 2,000 V; reflectron, 5,200 V; source, 15,000 V, with data acquisition using MassLynx software (ver. 4.0). These operating conditions provide mass resolution greater than 12,000.