Name: profiling the triacylglyceride contents in bat integumentary lipids by preparative thin layer chromatography and maldi-tof mass spectrometry
Uploaded: 2013-09-05T13:00:00
Duration: 9 min 18 s
Description: 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

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 border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Butylated Hydroxytoluene</td>
			<td>MP Biomedicals, LLC</td>
			<td>101162</td>
			<td><a href="http://www.mpbio.com" target="_blank">www.mpbio.com</a></td>
		</tr>
		<tr>
			<td>TLC Flexible Plates</td>
			<td>Whatman</td>
			<td>4410-222</td>
			<td><a href="http://www.whatman.com" target="_blank">www.whatman.com</a></td>
		</tr>
		<tr>
			<td>ACTH</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>A8346-5X1VL</td>
			<td><a href="http://www.sigmaaldrich.com" target="_blank">www.sigmaaldrich.com</a></td>
		</tr>
		<tr>
			<td>Triolein</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>44895-U</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC Lipid Standard</td>
			<td></td>
			<td>TLC 18-1</td>
			<td><a href="http://www.nu-chekprep.com" target="_blank">www.nu-chekprep.com</a></td>
		</tr>
		<tr>
			<td>MALDI TAG Standard</td>
			<td>Nu-Check Prep., Inc.</td>
			<td>NIH Code 53B</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI TAG Standard</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>17810-1AMP-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Vials with Teflon Cap</td>
			<td>U.S. National Scientific Co.</td>
			<td>B7800-2</td>
			<td><a href="http://www.nationalscientific.com/" target="_blank">www.nationalscientific.com/</a></td>
		</tr>
		<tr>
			<td>DHB</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>50862-1G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>CHCA</td>
			<td>Sigma-Aldrich Chem. Co.</td>
			<td>C8982-10X10 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Sebutape</td>
			<td>CuDerm</td>
			<td>S100</td>
			<td></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://www.jove.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://www.jove.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://www.jove.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 ...

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Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

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Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

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Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

Fabrication of Thin Film Silver/Silver Chloride Electrodes with Finely Controlled Single Layer Silver Chloride

This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin film silver electrodes. Thin film silver electrodes sized 80 &#181;m x 80 &#181;m and 160 &#181;m x 160 &#181;m were sputtered on quartz wafers with a chromium/gold (Cr/Au) layer for adhesion. After passivation, polishing and cathodic cleaning processes, the electrodes underwent galvanostatic oxidation with consideration of Faraday&#39;s Law of Electrolysis to form smooth layers of AgCl with a designated degree of coverage on top of the silver electrode. This protocol is validated by inspection of scanning electron microscope (SEM) images of the surface of the fabricated Ag/AgCl thin film electrodes, which highlights the functionality and performance of the protocol. Sub-optimally fabricated electrodes are fabricated as well for comparison. This protocol can be widely used to fabricate Ag/AgCl electrodes with specific impedance requirements (e.g., probing electrodes for impedance sensing applications like impedance flow cytometry and interdigitated electrode arrays).

This paper aims to present a method to form smooth and well-controlled films of silver chloride (AgCl) with designated coverage on top of thin film silver electrodes.

This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin ...

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. &#946;-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of ...