Name: analysis of the lipid composition of mycobacteria by thin layer chromatography
Uploaded: 2021-04-16T13:00:00.000+00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography at JoVE.com

This research was funded by the Spanish Ministry of Science, Innovation and Universities (RTI2018-098777-B-I00), the FEDER Funds, and the Generalitat of Catalunya (2017SGR-229). Sandra Guallar-Garrido is the recipient of a PhD contract (FI) from the Generalitat de Catalunya.

1. Extraction of the total non-covalent-linked lipids from mycobacteria (Figure 1)
<ol>
	<li>Scratch 0.2 g of mycobacteria from a solid media and add to a glass tube with a polytetrafluoroethlene (PTFE) liner screw caps. Add a solution consisting of 5 mL of chloroform and 10 mL of methanol (chloroform:methanol, 1:2). 
	NOTE: When organic solvents are used, only glass recipient should be used. No plastic containers are allowed. Moreover, PTFE liner screw caps for bottles are needed. 
	CAUTION: Chloroform is a potentially toxic and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Methanol is a potentially toxic and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).</li>
	<li>Leave the tube in constant stirring overnight to extract non-covalent-linked lipids from the mycobacterial cell surface. 
	NOTE: If an orbital shaking platform is not available, constant stirring can be replaced by periodic manual stirring as frequently as possible.</li>
	<li>Cover a glass funnel with a filter paper, filter the organic solvents, and collect them in a glass tube.</li>
	<li>Use a nitrogen gas flux to evaporate the liquid phase in the tube. Fill the tube with nitrogen gas, cover and store it at 4 &#176;C. 
	NOTE: Connect a glass Pasteur pipette to the stream of nitrogen gas to specifically evaporate the desired tube. Additionally, maintain the tube inside a dry block heater for tubes at 37 &#176;C. When the solvent evaporates, fill the tube with nitrogen gas before closing it.</li>
	<li>Add 15 mL of a solution of chloroform:methanol (2:1) to the cellular debris. Leave the tube in constant stirring overnight to extract non-covalent-linked lipids from the mycobacterial cell surface. 
	NOTE: If an orbital shaking platform is not available, constant stirring can be replaced by periodic manual stirring as frequently as possible.</li>
	<li>Let the mixture rest for 1 h. With a Pasteur pipette, recover the organic solvents. Cover a glass funnel with a filter paper and filter the organic solvents and collect them in the same glass tube previously used in step 1.3. Use a nitrogen gas flux to evaporate the liquid phase in the tube. Fill the tube with nitrogen gas, close it and store it again at 4 &#176;C.</li>
</ol>
2. Mycolic acid extraction by acid methanolysis (Figure 2A)
<ol>
	<li>Add 2-5 mL of esterifying solution into a hermetic glass tube with a PTFE liner screw cap. Add 0.2 g of mycobacteria biomass into the glass tube. 
	NOTE: Esterifying solution is formed by mixing 30 mL of methanol, 15 mL of toluene, and 1 mL of sulfuric acid. Mycobacteria cells can be taken from solid cultures or, even from delipidated cells after performing extraction of total non-covalent-linked lipids from mycobacteria (remaining cells after filtering in step 1.6). 
	CAUTION: Toluene is a flammable and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Sulfuric acid is a corrosive and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).</li>
	<li>Mix the content by vortexing. Let the mixture stand inside a dry bath at 80 &#176;C overnight.</li>
	<li>Allow the tube to cool until it reaches the room temperature and then add 2 mL of n-hexane to the tube. Mix the contents by vortexing for 30 s and allow the tube to settle until two clear phases appear. 
	CAUTION: n-hexane is a potential flammable, irritant, environmentally damaging, and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).</li>
	<li>Recover the upper phase corresponding to the n-hexane phase. Transfer it to a new tube.</li>
	<li>Repeat the step 2.3. Recover the upper phase again and transfer it to the same tube used in step 2.4.</li>
	<li>Evaporate the contents of the tube using a nitrogen gas flux. Fill the tube with nitrogen gas, close it, and store it at 4 &#176;C.</li>
</ol>
3. Mycolic acid extraction by saponification and methylation (Figure 2B)
<ol>
	<li>Scratch 0.2 g of mycobacteria from a solid media and add to a glass tube with a PTFE screw cap.</li>
	<li>Add 2 mL of methanol-benzene solution (80:20) containing 5% potassium hydroxide. Mix the contents by vortexing. Heat the mixture for 3 h at 100 &#176;C. 
	CAUTION: Benzene is a flammable, carcinogenic, and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).</li>
	<li>Allow the tube to cool to room temperature. Add 20% sulfuric acid to acidify the samples to achieve pH = 1.</li>
	<li>Add 3 mL of diethyl ether. Gently mix the contents by vortexing.</li>
	<li>Let the two phases form by settling. Recover the diethyl ether phase and transfer to a new tube. Repeat the wash step for a total of three times.</li>
	<li>Wash the diethyl ether extract with 2 mL of distilled water and transfer the upper part corresponding to the diethyl ether to a new tube. Repeat the wash step for a total of three times.</li>
	<li>Add 2 g of anhydrous sodium sulfate over the diethyl ether extract to dry it.</li>
	<li>Filter the suspension. Evaporate the content using a nitrogen gas flux.</li>
	<li>To perform the methylation step, dissolve 3 g of N-nitroso-N-methyl urea in a precooled solution formed by 45 mL of diethyl ether and 9 mL of 40% KOH in distilled water. 
	CAUTION: N-nitroso-N-methylurea is a toxic, irritant, carcinogenic, and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).</li>
	<li>Transfer the supernatant (diazomethane) to a new flask cooled in ice containing potassium hydroxide pellets (approximately 30 g). 
	NOTE: If the supernatant is not immediately used, it can be stored at -20 &#176;C for a maximum of 1 h. 
	CAUTION: Potassium hydroxide pellets are an irritant and corrosive substance. This material must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Diazomethane is highly toxic and potentially explosive. It must be used in a laminar flow hood with safety glass wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).</li>
	<li>Add 2 mL of the ether solution containing diazomethane, obtained in step 3.10, into the dried diethyl ether extract that contains mycolic acids, obtained in step 3.8. Incubate for 15 min at room temperature.</li>
	<li>Evaporate the suspension at 40 &#176;C. Fill the tube with nitrogen gas, close it, and store the methylated lipids at 4 &#176;C. 
	&#8203;NOTE: Evaporate the diazomethane from the ether solution under the laminar flow hood, until the ether loses the yellow color.</li>
</ol>
4. Thin layer chromatography (TLC) analysis
<ol>
	<li>Saturate the glass TLC chamber. To do this, cover one of the walls of the TLC chamber with a piece of filter paper and allow it to be in contact with the mobile phase composed by the mixture of solvents. Place the remaining volume of the solvent onto the bottom of the TLC chamber. 
	NOTE: The bottom of the TLC chamber must be covered by at least 1 cm of the mobile phase. In the present experiments, different mobile phases were used to develop the TLCs. They consisted of 85 mL of n-hexane plus 15 mL of diethyl ether; 100 mL of dichloromethane; 90 mL of chloroform, 10 mL of methanol, and 1 mL of water; 30 mL of chloroform, plus 8 mL of methanol, and 1 mL of water; 60 mL of chloroform, plus 35 mL of methanol, and 8 mL of water; 95 mL chloroform plus 5 mL of methanol; and 90 mL of petroleum ether (60-80 &#176;C) plus 10 mL of diethyl ether. 
	NOTE: In the two-dimensional TLC, use n-hexane:acetone (95:5) in the first direction three times, and use a single development with toluene:acetone (97:3) in the second direction to analyze mycolic acid composition. To analyze PIMs, use chloroform:methanol:water (60:30:6) in the first direction once, and use chloroform:acetic acid:methanol:water (40:25:3:6) in the second direction. To analyze PDIM and AG, use petroleum ether (60-80 &#176;C):ethyl acetate (98:2) in the first direction three times, and use a single development with petroleum ether (60-80 &#176;C):acetone (98:2) in the second direction. 
	CAUTION: Diethyl ether is a potentially toxic and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Dichloromethane is a potentially toxic and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Petroleum ether is a potential flammable, environmentally damaging and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Acetic acid is a potential flammable and corrosive substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Ethyl acetate is a flammable and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Acetone is a flammable and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).</li>
	<li>Close the TLC chamber to saturate it for at least 20 min. Meanwhile, dissolve the lipids present in the glass tube in 0.2-1 mL of chloroform. 
	NOTE: The volume used to dissolve the lipids can be modified depending on the desired or expected concentration of the sample.</li>
	<li>Apply 10 &#181;L of each suspension using a capillary glass tube directly on the TLC plate and let the sample dry for 5 min at room temperature. 
	NOTE: Samples must be applied at the bottom part of the plate leaving 1 cm on each side. Samples must be separated one from another for at least 0.5 cm. Once the sample is applied on the plate, tubes can be evaporated again with nitrogen gas and stored at 4 &#176;C for further use.</li>
	<li>Insert the plate into the saturated TLC chamber containing the mobile phase. Allow the mobile phase to run through the TLC. 
	NOTE: Any movement applied to the TLC chamber affects the running solvent on the plate and affects lipid mobility. In the case of performing two-dimensional TLC, two TLC chambers are required to contain both elution systems.</li>
	<li>Remove the plate from the TLC chamber when the solvent reaches 1 cm distance from the upper end of the plate. Leave the plate under laminar flux until the silica is totally dried. 
	NOTE: In the case of analyzing the mycolic acid composition, using n-hexane and diethyl-ether (85:15),&#160;repeat steps 4.4 and 4.5 two times more, until running the mobile phase three times over the TLC plate.</li>
	<li>Reveal the plate with the required stain; heat the plate if required. 
	NOTE: In the present experiment, 15-20 mL of the following solutions were used to spray the TLC plates: 10% Molybdatophosphoric acid hydrate in ethanol until the plate is bright yellow, followed by heating the plate at 120 &#176;C; 5% in ethanol of&#160;20% &#945;-naphthol in sulfuric acid followed by heating the plate at 120 &#176;C; Molybdenum Blue reagent (1.3% molybdenum oxide in 4.2 M sulfuric acid) until phosphate bands appeared or 1% anthrone in sulfuric acid. 
	CAUTION: Molybdatophosphoric acid hydrate is a flammable and corrosive substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Ethanol is a potential flammable and hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: 1-Naphthol is a flammable, corrosive, and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves). 
	CAUTION: Molybdenum Blue Spray Reagent is a corrosive, toxic, and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal protective equipment (laboratory coat, protective eyewear, and nitrile gloves).</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+lipids+of+Mycobacterium+tuberculosis.">Analysis of the lipids of Mycobacterium tuberculosis.</a> Mycobacterium tuberculosis Protocols. 54, 229-245 (2001).</li><li>Ojha, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Mycobacterium+tuberculosis+biofilms+containing+free+mycolic+acids+and+harbouring+drug-tolerant+bacteria.">Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.</a> Molecular Microbiology. 69 (1), 164-174 (2008).</li><li>Ojha, A. K., Trivelli, X., Guerardel, Y., Kremer, L., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+hydrolysis+of+trehalose+dimycolate+releases+free+mycolic+acids+during+mycobacterial+growth+in+biofilms.">Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms.</a> The Journal of Biological Chemistry. 285 (23), 17380-17389 (2010).</li><li>Layre, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycolic+acids+constitute+a+scaffold+for+mycobacterial+lipid+antigens+stimulating+CD1-restricted+T+cells.">Mycolic acids constitute a scaffold for mycobacterial lipid antigens stimulating CD1-restricted T cells.</a> Chemistry and Biology. 16 (1), 82-92 (2009).</li><li>Llorens-Fons, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+polyphleates,+external+cell+wall+lipids+in+Mycobacterium+abscessus,+are+associated+with+the+formation+of+clumps+with+cording+morphology,+which+have+been+associated+with+virulence.">Trehalose polyphleates, external cell wall lipids in Mycobacterium abscessus, are associated with the formation of clumps with cording morphology, which have been associated with virulence.</a> Frontiers in Microbiology. 8, (2017).</li><li>Butler, W. R., Guthertz, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycolic+acid+analysis+by+high-performance+liquid+chromatography+for+identification+of+mycobacterium+species.">Mycolic acid analysis by high-performance liquid chromatography for identification of mycobacterium species.</a> Clinical Microbiology Reviews. 14 (4), 704-726 (2001).</li><li>Teramoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mycolic+acids+in+total+fatty+acid+methyl+ester+fractions+from+Mycobacterium+species+by+high+resolution+MALDI-TOFMS.">Characterization of mycolic acids in total fatty acid methyl ester fractions from Mycobacterium species by high resolution MALDI-TOFMS.</a> Mass Spectrometry. 4 (1), 0035 (2015).</li><li>Sartain, M. J., Dick, D. L., Rithner, C. D., Crick, D. C., Belisle, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidomic+analyses+of+Mycobacterium+tuberculosis+based+on+accurate+mass+measurements+and+the+novel+"Mtb+LipidDB".">Lipidomic analyses of Mycobacterium tuberculosis based on accurate mass measurements and the novel "Mtb LipidDB".</a> Journal of Lipid Research. 52 (5), 861-872 (2011).</li><li>Li, M., Zhou, Z., Nie, H., Bai, Y., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+of+chromatography+and+mass+spectrometry+in+lipidomics.">Recent advances of chromatography and mass spectrometry in lipidomics.</a> Analytical and Bioanalytical Chemistry. 399 (1), 243-249 (2011).</li><li>Nahar, A., Baker, A. L., Nichols, D. S., Bowman, J. P., Britz, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Thin-Layer+Chromatography-Flame+Ionization+Detection+(TLC-FID)+to+total+lipid+quantitation+in+mycolic-acid+synthesizing+Rhodococcus+and+Williamsia+species.">Application of Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID) to total lipid quantitation in mycolic-acid synthesizing Rhodococcus and Williamsia species.</a> International Journal of Molecular Sciences. 21 (5), 1670 (2020).</li></ol>

The authors have nothing to disclose.

A simple protocol considered as the gold standard method for the extraction of noncovalently linked lipid compounds from the mycobacterial cell wall is presented. Further visualization by one- and two-dimensional TLCs from the extracted lipids of four different mycobacteria is shown.
Two consecutive combined mixtures of chloroform and methanol to recover the lipidic content of mycobacterial cells is the most widely used solvent mixture23,24<...

Mycobacterium is a genus that comprises pathogenic and non-pathogenic species, characterized by having a highly hydrophobic and impermeable cell wall formed by their peculiar lipids. Specifically, the mycobacterial cell wall contains mycolic acids, which are &#945;-alkyl and &#946;-hydroxy fatty acids, in which the &#945;-branch is constant in all mycolic acids (except for the length) and the &#946;-chain, called the meromycolate chain, is a long aliphatic chain that may contain different functional chemical groups described along with the literature (&#945;-, &#945;&#39;-, methoxy-, &#954;-, epoxy-, carboxy-, and &#969;-1-methoxy- mycolates), therefore producing seven types of mycolic acids (I-VII)1. Moreover, other lipids with unquestionable importance are also present in the cell wall of mycobacteria species. Pathogenic species such as Mycobacterium tuberculosis, the&#160;causative agent of tuberculosis2 produce specific lipid-based virulence factors such as phthiocerol dimycocerosates (PDIMs), phenolic glycolipid (PGL), di-, tri-, and penta-acyltrehaloses (DAT, TAT, and PAT), or sulfolipids, among others3. Their presence on the mycobacterial surface have been associated with the ability to modify the host immune response and therefore, the evolution and persistence of the mycobacterium inside the host4. For instance, the presence of triacylglycerols (TAG) has been associated with the hypervirulent phenotype of Lineage 2-Beijing sub-lineage of M. tuberculosis, possibly due to its capacity to attenuate the host immune response5,6. Other relevant lipids are lipooligosaccharides (LOSs) present in tuberculous and nontuberculous mycobacteria. In the case of Mycobacterium marinum, the presence of LOSs in its cell wall is related to sliding motility and the ability to form biofilms and interferes with recognition by macrophage pattern recognition receptors, affecting uptake and elimination of the bacteria by host phagocytes7,8. Additionally, the absence or presence of some lipids allows members of the same species to be classified into different morphotypes with virulent or attenuate profiles when interacting with host cells. For instance, the absence of glycopeptidolipids (GPL) in the rough morphotype of Mycobacterium abscessus has been associated with the ability to induce intraphagosomal acidification, and consequently cell apoptosis9, unlike the smooth morphotype that possesses GPLs in their surface. Furthermore, the lipid content of the mycobacterial cell wall is related to the ability to modify the immune response in the host. This is relevant in the context of using some mycobacteria to trigger a protective immune profile against different pathologies10,11,12,13.&#160;It has been demonstrated, for example, that Mycolicibacterium vaccae, a saprophytic mycobacterium, which is currently in phase III clinical trials as an immunotherapeutic vaccine for tuberculosis, display two colonial morphotypes. While the smooth phenotype, that contains a polyester in its surface, triggers a Th2 response, the rough phenotype devoid of the polyester can induce a Th1 profile when it interacts with host immune cells14. The repertoire of lipids present in the mycobacterial cell not only depends on mycobacteria species, but also on the conditions of mycobacterial cultures: time of incubation15,16 or composition of the culture medium17,18. In fact, changes in the culture medium composition affect the antitumor and immunostimulatory activity of M. bovis BCG&#160;and Mycolicibacterium brumae in vitro17. Moreover, the protective immune profile triggered by M. bovis BCG against M. tuberculosis challenge in mice models also depends on the culture media in which M. bovis BCG grows17. These could then be related to the lipid composition of the mycobacteria in each culture condition. For all these reasons, the study of the lipid content of mycobacteria is relevant. A visual procedure to extract and analyze the lipid composition of the mycobacterial cell wall is presented.

<table><tbody><tr><td>Acetic Acid</td><td>Merck</td><td>100063</td><td>CAUTION. Anhydrous for analysis EMSURE&amp;reg; ACS,ISO,Reag. Ph Eur</td></tr><tr><td>Acetone</td><td>Carlo Erba</td><td>400971N</td><td>CAUTION. ACETONE RPE-ACS-ISO FOR ANALYS ml 1000</td></tr><tr><td>Anthrone</td><td>Merck</td><td>8014610010</td><td>Anthrone for synthesis.</td></tr><tr><td>Benzene</td><td>Carlo Erba</td><td>426113</td><td>CAUTION. Benzene RPE - For analysis - ACS 2.5 l</td></tr><tr><td>Capillary glass tube</td><td>Merck</td><td>BR708709</td><td>BRAND&amp;reg;&amp;nbsp;disposable BLAUBRAND&amp;reg;&amp;nbsp;micropipettes, intraMark</td></tr><tr><td>Chloroform</td><td>Carlo Erba</td><td>412653</td><td>CAUTION. Chloroform RS - For HPLC - Isocratic grade - Stabilized with ethanol 2.5 L</td></tr><tr><td>Dry block heater</td><td>J.P. Selecta</td><td>7471200</td><td></td></tr><tr><td>Dicloromethane</td><td>Carlo Erba</td><td>412622</td><td>CAUTION. Dichloromethane RS - For HPLC - Isocratic grade - Stabilized with amylene 2.5 L</td></tr><tr><td>Diethyl ether</td><td>Carlo Erba</td><td>412672</td><td>CAUTION. Diethyl ether RS - For HPLC - Isocratic grade - Not stabilized 2.5 L</td></tr><tr><td>Ethyl Acetate</td><td>Panreac</td><td>1313181211</td><td>CAUTION. Ethyl acetate (Reag. USP, Ph. Eur.) for analysis, ACS, ISO</td></tr><tr><td>Ethyl Alcohol Absolute</td><td>Carlo Erba</td><td>4146072</td><td>CAUTION. Ethanol absolute anhydrous RPE - For analysis - ACS - Reag. Ph.Eur. - Reag. USP 1 L</td></tr><tr><td>Glass funnel</td><td>VidraFOC</td><td>DURA.2133148 1217/1</td><td></td></tr><tr><td>Glass tube</td><td>VidraFOC</td><td>VFOC.45066A-16125</td><td>Glass tube with PTFE recovered cap</td></tr><tr><td>Methanol</td><td>Carlo Erba</td><td>412722</td><td>CAUTION. Methanol RS - For HPLC - GOLD - Ultragradient grade 2.5 L</td></tr><tr><td>Molybdatophosphoric acid hydrate</td><td>Merck</td><td>51429-74-4</td><td>CAUTION.</td></tr><tr><td>Molybdenum Blue Spray Reagent, 1.3%</td><td>Sigma</td><td>M1942-100ML</td><td>CAUTION.</td></tr><tr><td>n-hexane</td><td>Carlo Erba</td><td>446903</td><td>CAUTION. n-Hexane 99% RS - ATRASOL - For traces analysis 2.5 L</td></tr><tr><td>n-nitroso-n-methylurea</td><td>Sigma</td><td>N4766</td><td>CAUTION</td></tr><tr><td>Orbital shaking platform</td><td>DDBiolab</td><td>995018</td><td>NB-205L benchtop shaking incubator</td></tr><tr><td>Petroleum ether (60-80&amp;ordm;C)</td><td>Carlo Erba</td><td>427003</td><td>CAUTION. Petroleum ether 60 - 80&amp;deg;C RPE - For analysis 2.5 L</td></tr><tr><td>Sprayer</td><td>VidraFOC</td><td>712/1</td><td></td></tr><tr><td>Sodium sulphate anhydrous</td><td>Merck</td><td>238597</td><td></td></tr><tr><td>Sulfuric acid 95-97%</td><td>Merck</td><td>1007311000</td><td>CAUTION. Sulfuric acid 95-97%</td></tr><tr><td>TLC chamber</td><td>Merck</td><td>Z204226-1EA</td><td>Rectangular TLC developing tanks, complete L &amp;times; H &amp;times; W 22 cm &amp;times; 22 cm &amp;times; 10 cm</td></tr><tr><td>TLC plate</td><td>Merck</td><td>1057210001</td><td>TLC SilicaGel 60- 20x20 cm x 25 u</td></tr><tr><td>TLC Plate Heater</td><td>CAMAG</td><td>223306</td><td>CAMAG TLC Plat Heater III</td></tr><tr><td>Toluene</td><td>Carlo Erba</td><td>488551</td><td>CAUTION. Toluene RPE - For analysis - ISO - ACS - Reag.Ph.Eur. - Reag.USP 1 L</td></tr><tr><td>Vortex</td><td>Fisher Scientific</td><td>10132562</td><td>IKA&amp;nbsp;Agitador IKA v&amp;oacute;rtex 3</td></tr><tr><td>1-naphthol</td><td>Sigma-Aldrich</td><td>102269427</td><td>CAUTION.</td></tr></tbody></table>

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.

With the aim of showing a wide range of lipids present in different mycobacteria species, M. bovis BCG was selected as it is rough and slow-growing mycobacteria. The rough and fast-growing M. fortuitum and M. brumae were added in the procedure and, finally, the smooth morphotype of M. abscessus was also included. These four species permit us to visualize a broad spectrum of mycobacteria-derived lipids such as acyltrehaloses (AT), GPLs, PDIM, PGL, PIM, TDM, and TMM. Moreover, all four s...

Watch this Scientific Journal Video about Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography at JoVE.com

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

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 ...

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7.8K Views.  Universitat Autònoma de Barcelona. 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.

Video: Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Chemistry

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.

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 ...

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

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

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

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Medicine

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Preparing Mtb Secretome to Study Host-Pathogen Interactions

Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, apoptosis, etc., to enhance survival. While the function of a few secretory factors has been elucidated, many are yet to be characterized. Hence, investigating the Mtb secretory protein is an essential area of research. Mtb secretes proteins into the extracytoplasmic space with the help of three different secretion systems: general, twin-arginine, and specialized Type VII secretion system. One has to prepare the secretome to identify novel secretory molecules and functionally characterize the known factors. Here, we outline the method for preparing the secretome and sample preparation for mass spectrometry. Secretome preparation requires the utmost care to avoid contamination of cytosolic proteins, which can be monitored by probing with antibodies against well-characterized membrane, secretory, and cytosolic proteins. The culture filtrate thus prepared can be used for different purposes, which include investigating host immunomodulation properties.

Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis

Here, we demonstrate the preparation of Mtb culture filtrate (CF), which is the bacterial growth media that consists of proteins secreted by the bacteria. Mtb secretes proteins to modulate host signaling and immune response, which are crucial for pathogenesis. The CFs prepared are analyzed by mass spectrometry to identify all the secretory factors.

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Summary

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Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

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

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis