Assistance was provided by Scott Treece, Katelyn Arter, Jeremy Ragsdell, Tony LaMark James, Amy Fischer, Hannah Blair, and Cheyenne Gerdes during the development of laboratory methods. We would like to thank the Medina-Bolivar Laboratory (Luis H. Nopo-Olazabal; Arkansas Biosciences Institute) for assistance with TLC scanning densitometry. MALDI-TOF MS used for this project was provided through the NSF EPSCoR, RII: Arkansas ASSET Initiative P3 Center (EPS-0701890) in the Arkansas Biosciences Institute. Funding was provided by a U.S. Fisheries and Wildlife Service/Arkansas State Wildlife Grant, the National Speleological Society, and The Center for North American Bat Research and Conservation at Indiana State University.

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&#39;s Institutional Biosafety Committee approval # 135349-1)28.
1. Tissue Treatment and Lipid Extraction
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>After 2 hr, add about 0.5 g anhydrous sulfate to each flask, mix briefly, and collect the solvent by filtering through filter paper.</li>
	<li>Repeat steps 1.2 and 1.3 twice. Once with 1:1 C:M and sequentially with 1:2 C:M. Pool filtrates together.</li>
	<li>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 &#176;C. It is generally best to analyze samples within one month after sample collection and to minimize freeze-thaw cycles.</li>
</ol>
2. Lipid Separation by Preparative Thin-layer Chromatography
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#956;l mixture of sterol, FFAs, TAGs, and sterol ester standards (use at 10 mg/ml; premade mixtures can be obtained).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Store the dried residue containing TAGs under N2 in the dark at 4 &#176;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.</li>
</ol>
3. TAG Analysis by MALDI-TOF MS
<ol>
	<li>Prepare a fresh &#945;-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).</li>
	<li>Prepare adrenocarticotropic hormone (ACTH; 18-39 clip, 2465.1989 Da) for instrument resolution and sensitivity testing by mixing 1 &#956;l of ACTH (1 mg/ml) with 39.5 &#956;l of 0.1% trifluoroacetic acid (TFA) to obtain a 10 pmol/&#956;l stock solution. This can be stored at -20 &#176;C for future use.</li>
	<li>Prepare a fresh working ACTH solution (1 pmol/&#956;l) by taking 1 &#956;l of the 10 pmol/&#956;l stock solution, mixing with 9 &#956;l 0.1% TFA, and then mixing (1:1) with 10 &#956;l of the CHCA matrix solution to obtain a 500 fmol/&#956;l concentration.</li>
	<li>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]).</li>
	<li>Prepare triolein at 10 mg/ml in 3:2 C:M for an external lock-mass calibration TAG standard.</li>
	<li>Dissolve the stored TAG samples in hexane to a 10 mg/ml solution.</li>
	<li>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.</li>
	<li>Mix (in pre-washed tubes) 10.0 &#956;l DHB matrix, 10.0 &#956;l sample or standard, and 5.0 &#956;l 1.0 M NaOH. Mix and briefly centrifuge the tube to bring mixture to the bottom.</li>
	<li>Spot 1.0 &#956;l 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.</li>
	<li>Perform MALDI-TOF MS analyses in positive reflectron mode. Tune and calibrate the instrument as described by the instrument&#39;s manufacturer using the ACTH and TAG standard solutions.</li>
	<li>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 (<a href="http://www.lipidmaps.org/tools/ms/glycerolipids_batch" target="_blank">www.lipidmaps.org/tools/ms/glycerolipids_batch</a>).</li>
	<li>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.</li>
</ol>
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&#39;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.

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The authors declare no competing financial interests.

This paper presents a simple and robust method for separating broad lipid classes isolated from mammalian integument by preparative TLC and determining TAG profiles by MALDI-TOF MS, without time-consuming derivatization of the lipid molecules. The critical steps in producing quality spectra of TAGs with MALDI-TOF MS include: 1) Successful extraction of the compound with minimal contamination or oxidation; 2) Sufficient separation and isolation by chromatography; and 3) High resolution and mass accuracy by MALDI-TOF MS.
This paper demonstrates the method by extracting and separating the neutral lipid fraction from the plagiopatagium of the Eastern red bat to obtain TAG MS profiles. While the present study used a bat species (Mammalia: Chiroptera) these methods can be extended to study integumentary lipids of any mammalian species. Bat integument is characterized by being predominantly cholesterol, with lower amounts of TAGs, FFAs, squalene, and sterol/wax esters. Sebum lipid ratios in bats differ from humans in that squalene is present in low amounts (as opposed to up to 16% in humans), whilst cholesterol occurs in larger ratios (1 - 7% in humans but 26 - 62% in bats)22,31. Human hairs contain about 3% TAGs, while ratios up to 28% are found in Eastern red bat hair. The extraction of lipid samples from bats is similar for other species. While in this study cotton balls soaked in solvent are used to remove sebum, one can also invert a vial or sample tube containing solvent onto the surface of the integument multiple times. Specialized tape products also provide alternative means to extract surface lipids32. A critical part of a proper lipid extraction is to minimize contamination from skin oils. This is easily accomplished by keeping a squeeze bottle with methanol and spraying all glassware and utensils, wiping utensils with a tissue between all samples and wearing examination gloves. Oxidation of polyunsaturated acyls is prevented through the addition of BHT, and it should be used regardless of the temperature samples are stored at.
Biomolecule analysis usually requires a chromatographic step to separate molecules of interest from contaminants. TLC is used in this method, which avoids instrumentation requirements for gas or liquid chromatography and requires less technical experience to achieve robust and repeatable results. Depending on the lipid class of interest, many different mobile phases can be incorporated. Furthermore, the use of HPTLC plates and scanning densitometry can be used to achieve quantitative results. While the variations of TLC methods are too numerous to list here, some common mobile phases used in lipid TLC include chloroform:methanol:water for separation of phospholipids and glycolipids or isooctane:ethyl ether for separation of non-polar lipids28. In terms of separating TAGs from other lipid classes, the H:E:A solvent system works consistently and provides comparable results.
Another advantage for using TLC is that bands of interest can be rapidly profiled using MALDI-TOF MS without prior derivatization of the analytes. In this study the silica is removed from the TLC plate, and the analyte is eluted from it by sonication in a solvent and subsequent centrifugation to separate the adsorbent and evaporation of the eluting solvent. Alternatively, matrix may be applied directly onto analyte bands separated on TLC plates and then directly analyzed by MALDI-TOF MS33. Successful profiling by MALDI-TOF MS does rely on sufficient sample preparation and the operator&#39;s proficiency with tuning and calibrating the instrument. The instrument should be calibrated daily with standards covering molecular weight range appropriate for the molecules of interest. The suitable sensitivity and resolution (e.g. &#8805;10,000) of the equipment should also be confirmed daily with ACTH.
The sebaceous lipid constituents found on the surface of mammalian integument may play a role in colonization by bacterial/fungal pathogens. Therefore knowledge of the chemical composition among species and individuals may provide clues about processes of human and wildlife diseases. Intraspecific differences among diseased and healthy individuals may represent clinical signs that aid in disease detection and diagnosis. Furthermore, if specific compounds that inhibit microbial growth are present, these may be identified for use in disease treatment and prevention.

Mammalian integumentary tissues include the epidermis, keratinous structures (e.g. hair and nails), and exocrine glands. Sebaceous-type exocrine glands are associated with hair follicles, which are collectively referred to as the pilosebaceous unit1. Sebaceous glands release an oily exudate onto the skin surface referred to as sebum. Sebum is composed largely of glycerolipids (e.g. triacylglycerides [TAGs]), free fatty acyls (FFAs), sterol/wax esters, and squalene. Sebum&#39;s chemical composition is species-specific2. In addition to being part of the innate immune system and providing antimicrobial function3, sebaceous lipids affect important physiological processes including evaporative water loss through skin4, cellular integrity and gene regulation5, and drug absorption6. Sebaceous lipid compositions can also serve as disease markers. Altered ratios and amounts of sebaceous broad lipid classes are clinical signs of diseases such as acne vulgaris7, dandruff8, seborrheic dermatitis8, asteatosis9, among others10. Epidermal and hair tissues include variable profiles containing sterol and derivatives, TAGs, FFAs, ceramides, phospholipids, and other minor lipid components. Because integumentary lipids can function in disease processes, determining differences in chemical compositions of TAGs between healthy and diseased individuals may be useful for clinical diagnosis of disease.
Lipids are generally defined as being water-insoluble organic compounds with either nonpolar or nonpolar-polar substituents11. Lipid structures may be long hydrocarbon chains, oxygenated alkanes (including wax esters, FFAs, alcohols, ketones, and aldehydes), or complex ring structures such as cholesterol12. There are eight major classes of lipids based on structure (FFAs, glycerolipids [GL], glycerophospholipids [GP], sphingolipids [SP], sterol lipids [ST], prenol lipids [PR], saccharolipids [SL], and polyketides [PK]), which exhibit a wide range of chemical properties depending on the class13. Due to the wide variation in the chemical properties of lipid classes, direct profiling without prior derivatization of lipid molecules is desired. One emergent method in lipid research is thin-layer chromatography (TLC) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)14.
MALDI-TOF MS is used extensively in proteomic research to identify proteins and associate them to specific amino acid sequences due to the highly accurate peptide ion mass &#34;fingerprints&#34; generated from trypsin-digested proteins15. MALDI-TOF MS can also be used to profile other biomolecule classes, including lipids such as TAGs16-18. MALDI requires the use of a matrix, typically an organic compound that contains aromatic and conjugated double bond structures. The matrix molecules serve to gently transfer the energy of the laser to the analytes, promote proton transfer, and produce singly charged gas-phase ions19-21. Ions are subjected to a high voltage field under high vacuum and accelerated into a TOF mass analyzer where ions are subsequently separated by differences in velocities that are proportional to their mass-to-charge ratios. Even very large biomolecules can be ionized with little fragmentation, producing singly charged molecular ion species for simplified spectrum analysis. The ability to analyze lipid molecules directly without prior derivatization has promoted ready adoption of MALDI-TOF MS in lipidomic research18.
This paper presents a routine method to isolate and analyze integumentary lipids from the hair, sebaceous secretions and plagiopatagium of the Eastern red bat (Lasiurus borealis). This is used to determine interspecific variations in bat integumentary lipids to elucidate the disease process of White-Nose Syndrome (WNS)22. WNS is a fungal disease of bats and is caused by the newly described psychrophilic species Geomyces destructans23-25. WNS has caused the death of over 5 million North American cave bats and threatens the extinction of vulnerable bat species, with potential economic impacts of billions of dollars in damage to the agricultural industry26,27. To investigate the steps in G. destructans infection, lipids were extracted from the hair and wing tissues of Eastern red bats and separated into broad lipid classes by TLC to isolate the TAG fraction for subsequent analysis by MALDI-TOF MS. TAGs contain short acyl chains and are easily detected by MALDI-TOF MS with little matrix interference.

<table><tbody><tr><td>Butylated Hydroxytoluene</td><td>MP Biomedicals, LLC</td><td>101162</td><td>&lt;a href=&quot;http://www.mpbio.com&quot; target=&quot;_blank&quot;&gt;www.mpbio.com&lt;/a&gt;</td></tr><tr><td>TLC Flexible Plates</td><td>Whatman</td><td>4410-222</td><td>&lt;a href=&quot;http://www.whatman.com&quot; target=&quot;_blank&quot;&gt;www.whatman.com&lt;/a&gt;</td></tr><tr><td>ACTH</td><td>Sigma-Aldrich Chem. Co.</td><td>A8346-5X1VL</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com&quot; target=&quot;_blank&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>Triolein</td><td>Sigma-Aldrich Chem. Co.</td><td>44895-U</td><td>&amp;nbsp;</td></tr><tr><td>TLC Lipid Standard</td><td></td><td>TLC 18-1</td><td>&lt;a href=&quot;http://www.nu-chekprep.com&quot; target=&quot;_blank&quot;&gt;www.nu-chekprep.com&lt;/a&gt;</td></tr><tr><td>MALDI TAG Standard</td><td>Nu-Check Prep., Inc.</td><td>NIH Code 53B</td><td>&amp;nbsp;</td></tr><tr><td>MALDI TAG Standard</td><td>Sigma-Aldrich Chem. Co.</td><td>17810-1AMP-S</td><td>&amp;nbsp;</td></tr><tr><td>Glass Vials with Teflon Cap</td><td>U.S. National Scientific Co.</td><td>B7800-2</td><td>&lt;a href=&quot;http://www.nationalscientific.com/&quot; target=&quot;_blank&quot;&gt;www.nationalscientific.com/&lt;/a&gt;</td></tr><tr><td>DHB</td><td>Sigma-Aldrich Chem. Co.</td><td>50862-1G-F</td><td>&amp;nbsp;</td></tr><tr><td>CHCA</td><td>Sigma-Aldrich Chem. Co.</td><td>C8982-10X10 mg</td><td>&amp;nbsp;</td></tr><tr><td>Sebutape</td><td>CuDerm</td><td>S100</td><td>&amp;nbsp;</td></tr></tbody></table>

profiling the triacylglyceride contents in bat integumentary lipids by preparative thin layer chromatography and maldi-tof mass spectrometry

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

The extraction method for isolating total lipids from tissue described by Folch30 is a straightforward procedure, which is adapted here. After tissue extraction and solvent evaporation, the lipids often appear as a yellowish film. The yellow color most likely is from protein contaminants, which can be removed by performing a liquid-liquid extraction. This additional sample processing is not needed in this procedure because preparative TLC separates the TAG fraction from such contaminants. The addition of fresh anhydrous sodium sulfate at all filtering stages helps reduce water contamination that affects accurate lipid weight determinations.
Mammalian integumentary lipid analyses by preparative TLC with H:E:A as the mobile phase will usually resolve four distinct bands corresponding to (starting from origin) sterols, FFAs, TAGs, and sterol esters/wax esters/squalene (Figure 1). On occasion when using analytical high performance (HP) TLC with the H:E:A mobile phase, the sterol esters, waxy esters, and squalene will separate and appear as three separate bands. Under the conditions used in the present study, the sterol/waxy esters are not separated. If these bands are of interest, the mobile phase can be switched to isooctane:ethyl ether (95:5 v/v), and the HPTLC plate can be analyzed by scanning densitometry. Other factors can cause poor separation. These are usually eliminated by placing the filter paper in the chamber, applying grease for a tight seal on the lid, equilibrating the chamber overnight, and keeping clean TLC chambers, to obtain consistent quality TLC separations and data.
Representative MALDI-TOF mass spectra obtained for TAGs isolated from the Eastern red bat are shown in Figures 2 and 3. These spectra contain TAG ion peaks in the mass range between m/z 850 - 910, which is typical for TAGs isolated from non-aquatic mammals. The addition of 1.0 M NaOH promotes singly charged Na+ ions that are more stable than H+ ions. In addition to stability, the absence of H+ and K+ ions increases ease of spectrum analysis. The m/z 850 - 910 ion peaks correspond to 16:0, 18:0, 18:1, and 18:2 FA moieties being the dominant acyl constituents in TAGs (Table 1). Possible FA moieties of TAGs can be initially determined by total ion m/z present in MALDI-TOF MS spectra, and differences among species and individuals deduced. However, if the specific ratios of acyl content are required, then MS/MS or gas chromatography (GC/MS) must be used. Further information on acyl ratios can be deduced by observing the peaks in the diacylglycerides region of the spectrum (Figure 4). Diacylglycerides are produced from TAG fragmentation in the MALDI source and can be found in the m/z 590 - 650 region. TAG fragmentation can be increased by omitting the addition of 1.0 M NaOH16. Eastern red bat wing tissue is characterized by a dominant peak at m/z 879.7 and hair tissue with a dominant peak at m/z 881.8 (Figure 2 and 3 respectively). Peaks at m/z 907.8, 879.7, and 855.7 (POP, PPoS) are approximately even in intensity (~50%) in hair tissue with the peak at 853.7 being ~40%.
<table border="1">
	<tbody>
		<tr>
			<td fo:width="2in">Composition</td>
			<td fo:width="1in">Elemental Composition</td>
			<td fo:width="1in">Observed Mass</td>
		</tr>
		<tr>
			<td>Na+TAGs</td>
			<td> </td>
			<td>Na+TAGs</td>
		</tr>
		<tr>
			<td>SSO</td>
			<td>C57H108O6</td>
			<td>911.8</td>
		</tr>
		<tr>
			<td>OOS, LSS</td>
			<td>C57H106O6</td>
			<td>909.8</td>
		</tr>
		<tr>
			<td>OOO, LnSS, LSO</td>
			<td>C57H104O6</td>
			<td>907.8</td>
		</tr>
		<tr>
			<td>LOO, LLS</td>
			<td>C57H102O6</td>
			<td>905.8</td>
		</tr>
		<tr>
			<td>LLO, OOLn</td>
			<td>C57H100O6</td>
			<td>903.7</td>
		</tr>
		<tr>
			<td>LLL</td>
			<td>C57H98O6</td>
			<td>901.7</td>
		</tr>
		<tr>
			<td>LLLn</td>
			<td>C57H96O6</td>
			<td>899.7</td>
		</tr>
		<tr>
			<td>LLnLn</td>
			<td>C57H94O6</td>
			<td>897.7</td>
		</tr>
		<tr>
			<td>LnLnLn</td>
			<td>C57H92O6</td>
			<td>895.7</td>
		</tr>
		<tr>
			<td>OSP</td>
			<td>C55H104O6</td>
			<td>883.8</td>
		</tr>
		<tr>
			<td>LSP, OOP, SOPo</td>
			<td>C55H102O6</td>
			<td>881.8</td>
		</tr>
		<tr>
			<td>LOP, LnSP, LSPo</td>
			<td>C55H100O6</td>
			<td>879.7</td>
		</tr>
		<tr>
			<td>LLP, LnOP, LOPo</td>
			<td>C55H98O6</td>
			<td>877.7</td>
		</tr>
		<tr>
			<td>LnLP, LLPo, LnOPo</td>
			<td>C55H96O6</td>
			<td>875.7</td>
		</tr>
		<tr>
			<td>LnLnP, LnLPo</td>
			<td>C55H94O6</td>
			<td>873.7</td>
		</tr>
		<tr>
			<td>LnLnPo</td>
			<td>C55H92O6</td>
			<td>871.7</td>
		</tr>
		<tr>
			<td>PPS</td>
			<td>C53H102O6</td>
			<td>857.8</td>
		</tr>
		<tr>
			<td>POP, PPoS</td>
			<td>C53H100O6</td>
			<td>855.7</td>
		</tr>
		<tr>
			<td>OOM, PPL, PoPoS, POPo</td>
			<td>C53H98O6</td>
			<td>853.7</td>
		</tr>
		<tr>
			<td>PPLn, PPoL, PoPoO, MyOO</td>
			<td>C53H96O6</td>
			<td>851.7</td>
		</tr>
		<tr>
			<td>LLM, LnOM</td>
			<td>C53H94O6</td>
			<td>849.7</td>
		</tr>
		<tr>
			<td>PPP, SSLa</td>
			<td>C51H98O6</td>
			<td>829.7</td>
		</tr>
		<tr>
			<td>PPPo, OSLa</td>
			<td>C51H96O6</td>
			<td>827.7</td>
		</tr>
		<tr>
			<td>PPoPo, PMyO</td>
			<td>C51H94O6</td>
			<td>825.7</td>
		</tr>
		<tr>
			<td>LnLnLa</td>
			<td>C51H86O6</td>
			<td>817.6</td>
		</tr>
		<tr>
			<td>MMS, SLaP, PPM</td>
			<td>C49H94O6</td>
			<td>801.7</td>
		</tr>
		<tr>
			<td>SLaPo, PPoM, PPMy</td>
			<td>C49H92O6</td>
			<td>799.7</td>
		</tr>
		<tr>
			<td>PoPoM, OOCa</td>
			<td>C49H90O6</td>
			<td>797.7</td>
		</tr>
		<tr>
			<td>MMP</td>
			<td>C47H90O6</td>
			<td>773.7</td>
		</tr>
		<tr>
			<td>MMPo, OCaP</td>
			<td>C47H88O6</td>
			<td>771.7</td>
		</tr>
		<tr>
			<td>OCaPo</td>
			<td>C47H86O6</td>
			<td>769.6</td>
		</tr>
		<tr>
			<td>MMM, PPCa, PMLa</td>
			<td>C45H86O6</td>
			<td>745.6</td>
		</tr>
		<tr>
			<td>PoPCa, PoMLa</td>
			<td>C45H84O6</td>
			<td>743.6</td>
		</tr>
		<tr>
			<td>PoPoCa</td>
			<td>C45H82O6</td>
			<td>741.6</td>
		</tr>
		<tr>
			<td>LaLaP, MMLa, MCaP</td>
			<td>C43H82O6</td>
			<td>717.6</td>
		</tr>
		<tr>
			<td>LaLaPo</td>
			<td>C43H80O6</td>
			<td>715.6</td>
		</tr>
		<tr>
			<td>OO</td>
			<td>C39H72O5</td>
			<td>643.5</td>
		</tr>
		<tr>
			<td>OL</td>
			<td>C39H70O5</td>
			<td>641.5</td>
		</tr>
		<tr>
			<td>LL</td>
			<td>C39H68O5</td>
			<td>639.5</td>
		</tr>
		<tr>
			<td>SP</td>
			<td>C37H72O5</td>
			<td>619.5</td>
		</tr>
		<tr>
			<td>OP</td>
			<td>C37H70O5</td>
			<td>617.5</td>
		</tr>
		<tr>
			<td>LP</td>
			<td>C37H68O5</td>
			<td>615.5</td>
		</tr>
	</tbody>
</table>
Table 1. Fatty acid composition, elemental composition, and isotopic mass of sodiated adducts of triacylglycerides and diacylglycerides. Ln=linolenic acid (18:3), L=linoleic acid (18:2), O=oleic acid (18:1), S=stearic acid (18:0), P=palmitic acid (16:0), Po=palmitoleic acid (16:1), M=myristic acid (14:0), My=myristoleic acid (14:1) La=lauric acid (12:0), Ca=Capric acid (10:0).
<img alt="Figure 1" src="/files/ftp_upload/50757/50757fig1.jpg" /> 
Figure 1. Thin-layer chromatogram of broad lipid class separation by hexane:diethyl ether:acetic acid (80:20:2 v/v/v) as the mobile phase. The band between sterol and FFA was not identified by a standard but may be a fatty alcohol or wax diester.
<img alt="Figure 2" src="/files/ftp_upload/50757/50757fig2.jpg" /> 
Figure 2. Expanded TAG region of MALDI-TOF mass spectrum of sodiated TAGs (m/z 700 - 950) from Eastern red bat (L. borealis) wing tissue. Peaks identified at m/z 853.7 (OOM, PPL, PoPoS, POPo) and m/z 879.7 (LOP, LnSP, LSPo). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50757/50757fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/50757/50757fig3.jpg" /> 
Figure 3. Expanded TAG region of MALDI-TOF mass spectrum of sodiated TAGs (m/z 700 - 950) from Eastern red bat (L. borealis) hair tissue. Peaks identified at m/z 905.8 (LOO, LLS) and m/z 907.8 (OOO, LnSS, LSO). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50757/50757fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/50757/50757fig4.jpg" /> 
Figure 4. DAG region of MALDI-TOF mass spectrum of sodiated DAG fragments (m/z 530 - 730) from Eastern red bat (L. borealis) wing tissue. Peaks identified at m/z 643.5 (OO) and m/z 615.5 (LP). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50757/50757fig4large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry at JoVE.com

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

Mammalian integument contains solvent-extractable lipids that can provide chemical compositions characteristic of individual species. This paper presents a routine method for separating broad lipid classes isolated from integumentary tissues using thin layer chromatography and determining the triacylglyceride profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune ...

profiling-triacylglyceride-contents-bat-integumentary-lipids

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

21.1K Views.  Arkansas State University. Mammalian integument contains solvent-extractable lipids that can provide chemical compositions characteristic of individual species. This paper presents a routine method for separating broad lipid classes isolated from integumentary tissues using thin layer chromatography and determining the triacylglyceride profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Biochemistry

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Environment

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial role in the functioning of the inner membrane. Its metabolite monolysocardiolipin (MLCL) is physiologically nearly absent in the lipid extract of animal cells and its appearance is the hallmark of the Barth syndrome (BTHS), a rare and often misdiagnosed genetic disease that causes severe cardiomyopathy in infancy. The method described here generates a "cardiolipin fingerprint" and allows a simple assay of the relative levels of CL and MLCL species in cellular lipid profiles. In the case of leukocytes, only 1 mL of blood is required to measure the MLCL/CL ratio via matrix-assisted laser desorption ionization - time-of-flight/mass spectrometry (MALDI-TOF/MS) just within 2 h from blood withdrawal. The assay is straightforward and can be easily integrated into the routine work of a clinical biochemistry laboratory to screen for BTHS. The test shows 100% sensitivity and specificity for BTHS, making it a suitable diagnostic test.

This protocol shows how to obtain a mass spectrometric "fingerprint" of leukocyte cardiolipin for the diagnosis of Barth syndrome. The assessment of elevated monolysocardiolipin to cardiolipin ratio discriminates patients with Barth syndrome from control heart failure patients with 100% sensitivity and specificity.

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial ...

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Screening Lipid Structures Using Stable Isotope Labeling

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (&lt;1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Quantification of Complex Lipidomic Samples Using Stable Isotope Labeling

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Biology

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

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

Summary

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Chapters in this video

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Fatty Acid ¹³C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

Shotgun Lipidomics of Rodent Tissues

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography