We are grateful to current and former members of the Titorenko laboratory for discussions. We acknowledge the Centre for Biological Applications of Mass Spectrometry and the Centre for Structural and Functional Genomics (both at Concordia University) for outstanding services. This study was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada (RGPIN 2014-04482) and Concordia University Chair Fund (CC0113). K.M. was supported by the Concordia University Merit Award.

1. Preparation of sterile media for culturing yeast
<ol>
	<li>Prepare 90 mL of a complete YP medium that contains 1% (w/v) yeast extract and 2% (w/v) bactopeptone.</li>
	<li>Prepare 90 mL of a synthetic minimal YNB medium containing 0.67% (w/v) yeast nitrogen base without amino acids, 20 mg/L L-histidine, 30 mg/L L-leucine, 30 mg/L L-lysine, and 20 mg/L uracil.</li>
	<li>Divide 90 mL of the complete YP medium equally into two 250 mL Erlenmeyer flasks (i.e., 45 mL each).</li>
	<li>Divide 90 mL...

<ol>
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Molecular and Cell Biology of Lipids. 1862 (1), 69-80 (2017).</li><li>Csord&#225;s, G., Weaver, D., Hajn&#243;czky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-mitochondrial+contactology:+structure+and+signaling+functions.">Endoplasmic reticulum-mitochondrial contactology: structure and signaling functions.</a> Trends in Cell Biology. 28 (7), 523-540 (2018).</li><li>Mitrofanova, D., 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=Lipid+metabolism+and+transport+define+longevity+of+the+yeast+Saccharomyces+cerevisiae.">Lipid metabolism and transport define longevity of the yeast Saccharomyces cerevisiae.</a> Frontiers in Bioscience (Landmark Edition). 23, 1166-1194 (2018).</li><li>Tamura, Y., Kawano, S., Endo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+contact+zones+as+sites+for+lipid+transfer.">Organelle contact zones as sites for lipid transfer.</a> Journal of Biochemistry. 165 (2), 115-123 (2019).</li><li>Weissman, J., Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to Yeast Genetics: Functional Genomics, Proteomics, and Other Systems Analyses. , (2010).</li><li>Botstein, D., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast:+an+experimental+organism+for+21st+Century+biology.">Yeast: an experimental organism for 21st Century biology.</a> Genetics. 189 (3), 695-704 (2011).</li><li>Duina, A. A., Miller, M. E., Keeney, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Budding+yeast+for+budding+geneticists:+a+primer+on+the+Saccharomyces+cerevisiae+model+system.">Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.</a> Genetics. 197 (1), 33-48 (2014).</li><li>Strynatka, K. A., Gurrola-Gal, M. C., Berman, J. N., McMaster, C. 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The authors declare that they have no competing financial interests.

The following precautions are important for the successful implementation of the protocol described here:
1. Chloroform and methanol are toxic. They efficiently extract various substances from surfaces, including laboratory plasticware and your skin. Therefore, handle these organic solvents with caution by avoiding the use of plastics in steps that involve contact with chloroform and/or methanol, using borosilicate glass pipettes for these steps, and rinsing these pipettes with chloroform and ...

A body of evidence indicates that lipids, one of the major classes of biomolecules, play essential roles in many vital processes within a eukaryotic cell. These processes include the assembly of lipid bilayers that constitute the plasma membrane and membranes surrounding cellular organelles, transport of small molecules across cell membranes, response to changes in the extracellular environment and intracellular signal transduction, generation and storage of energy, import and export of proteins confined to different organelles, vesicular trafficking of proteins within the endomembrane system and protein secretion, and several modes of regulated cell death1,2,3,4,5,6,7,8,9,10.
The budding yeast S. cerevisiae, a unicellular eukaryotic organism, has been successfully used to uncover some of the mechanisms underlying the essential roles of lipids in these vital cellular processes4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20. S. cerevisiae is a valuable model organism for uncovering these mechanisms because it is amenable to comprehensive biochemical, genetic, cell biological, chemical biological, system biological, and microfluidic dissection analyses21,22,23,24,25. Further progress in understanding mechanisms through which lipid metabolism and intracellular transport contribute to these vital cellular processes requires sensitive mass spectrometry technologies for the quantitative characterization of the cellular lipidome, understanding the lipidome molecular complexity, and integrating quantitative lipidomics into a multidisciplinary platform of systems biology1,2,3,26,27,28,29,30.
Current methods for the mass spectrometry-assisted quantitative lipidomics of yeast cells and cells of other eukaryotic organisms are not sufficiently versatile, robust, or sensitive. Moreover, these currently used methods are unable to differentiate various isobaric or isomeric lipid species from each other. Here we describe a versatile, robust, and sensitive method that allows use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantitative analysis of major cellular lipids of S. cerevisiae.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL High-speed glass centrifuge tubes with Teflon lined caps</td>
			<td>PYREX</td>
			<td>05-550</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Glass sample vials with Teflon lined caps</td>
			<td>Fisher Scientific</td>
			<td>60180A-SV9-1P</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A461-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A9554</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1100 series LC system</td>
			<td>Agilent Technologies</td>
			<td>G1312A</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent1100 Wellplate</td>
			<td>Agilent Technologies</td>
			<td>G1367A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Fisher Scientific</td>
			<td>A11450</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>A11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>A470-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Bactopeptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiolipin</td>
			<td>Avanti Polar Lipids</td>
			<td>750332</td>
			<td></td>
		</tr>
		<tr>
			<td>Centra CL2 clinical centrifuge</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramide</td>
			<td>Avanti Polar Lipids</td>
			<td>860517</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C297-4</td>
			<td></td>
		</tr>
		<tr>
			<td>CSH C18 VanGuard</td>
			<td>Waters</td>
			<td>186006944</td>
			<td>Pre-column system</td>
		</tr>
		<tr>
			<td>Free fatty acid (19:0)</td>
			<td>Matreya</td>
			<td>1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads (acid-washed, 425-600 &#956;M)</td>
			<td>Sigma-Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
		<tr>
			<td>L-histidine</td>
			<td>Sigma</td>
			<td>H8125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid Search software (V4.1)</td>
			<td>Fisher Scientific</td>
			<td>V4.1</td>
			<td>LC-MS/MS analysis software</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma</td>
			<td>L8912</td>
			<td></td>
		</tr>
		<tr>
			<td>L-lysine</td>
			<td>Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A4564</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850340</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850704</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylglycerol</td>
			<td>Avanti Polar Lipids</td>
			<td>840446</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylinositol</td>
			<td>Avanti Polar Lipids</td>
			<td>LM1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylserine</td>
			<td>Avanti Polar Lipids</td>
			<td>840028</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse-phase column CSH C18</td>
			<td>Waters</td>
			<td>186006102</td>
			<td></td>
		</tr>
		<tr>
			<td>Sphingosine</td>
			<td>Avanti Polar Lipids</td>
			<td>860669</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Orbitrap Velos MS</td>
			<td>Fisher Scientific</td>
			<td>ETD-10600</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricylglycerol</td>
			<td>Larodan, Malmo</td>
			<td>TAG Mixed FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic sonicator</td>
			<td>Fisher Scientific</td>
			<td>15337416</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast nitrogen base without amino acids</td>
			<td>Fisher Scientific</td>
			<td>DF0919-15-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast strain BY4742</td>
			<td>Dharmacon</td>
			<td>YSC1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Our method for a quantitative assessment of major cellular lipids within a yeast cell with the help of LC-MS/MS was versatile and robust. It allowed us to identify and quantify 10 different lipid classes in S. cerevisiae cells cultured in the synthetic minimal YNB medium initially containing 2% glucose. These lipid classes include free (unesterified) fatty acids (FFA), CL, phytoceramide (PHC), phytosphingosine (PHS), PC, PE, PG, PI, PS, and TAG (Supplemental Table 1</stro...

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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.

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 ...

This protocol provides an accurate and reproducible method for quantifying ten different lipid classes of Saccharomyces cerevisiae using a single extraction method and a single LC-MS method. This method allows the detection and quantification of isobaric and isomeric lipidic species having different chemical and physical properties. For Saccharomyces cerevisiae culture, first add 5 ml of sterile 20%glucose stock solution to each of two Erlenmeyer flasks containing sterile YNB medium to a final concentration of 2%glucose.Then, use a sterile pipette to transfer a volume of an overnight yeast culture containing five times ten to the seventh yeast cells into each flask and culture the flasks for at least 24 hours at 30 degrees Celsius with rotational shaking at 200 revolutions per minute. For lipid extraction, determine the total number of yeast cells per milliliter of culture and transfer five times ten to the seventh cells into a 15 ml centrifuge tube. Add ice cold nano-pure water to bring the total volume up to 15 ml and collect the cells by centrifugation.Re-suspend the yeast pellet in 15 ml of ice cold iso-tonic ammonium bicarbonate buffer for a second centrifugation and re-suspend the pellet in 1.5 ml of fresh ice cold ammonium bicarbonate solution. At the end of the centrifugation, store the pellet at 80 degrees celsius until the extraction. To begin the lipid extraction, thaw the cell pellet on ice before re-suspending the cells in 200 microliters of ice cold nano-pure water.In a fume hood, transfer the cell suspension to a 15 ml, high strength glass, screw top centrifuge tube with a polytetrafluoroethylene lined cap and add 25 microliters of an internal lipids standards mixture, prepared in 2:1 chloroform methanol. 100 microliters of 425 to 600 micromolar acid washed glass beads and 600 microliters of a 17:1 chloroform methanol to the tube. Vortex the tube at high speed for five minutes at room temperature to disrupt the cells.Followed by vortexing for one hour at low speed at room temperature to facilitate lipid extraction. At the end of the extraction, incubate the sample for 15 minutes on ice, to promote protein precipitation and the separation of the aqueous and organic phases. At the end of the incubation, centrifuge the sample to further separate the upper aqueous phase from the lower organic phase and, in the fume hood, use a borosilicate glass pipette to transfer the lower organic phase to a new 15 ml high strength glass screw top centrifuge tube with a polytetrafluoroethylene lined cap.Place the lower organic phase under the flow of nitrogen gas and, in the fume hood, add 300 microliters of a 2:1 chloroform methanol mixture to the remaining upper aqueous phase. To facilitate phosphatidicacid, phosphatidylserine, phosphatidylinositol and cardio-lipid extraction vortex the tube vigorously for five minutes at room temperature before centrifuging the sample. Use a borosilicate glass pipette to transfer the lower organic phase into the lower organic phase under nitrogen and evaporate the solvent in the combined organic phases in the fume hood.Then, close the tubes of lipid film under the flow of nitrogen gas and store the samples at 80 degrees celsius. For liquid chromatography, separation of the extracted lipids at 500 microliters of a 63:35:5 aceto-nitrile-2-propanol nano-pure water mixture to the tube of lipid film and vortex three times for ten seconds per vortex at room temperature. Subject the tube contents to ultrasonic sonication for 15 minutes before vortexing the tube three times for ten seconds per vortex as just demonstrated.Transfer 100 microliters of the sample into a glass vial with an insert used for a 24 well plate and eliminate any bubbles within the insert. Place the insert into one well of a 24 well plate and load a reverse phase C18 column CSH coupled to a pre-column into the liquid chromatography system. Set the column temperature to 55 degrees celsius and the flow to 300 microliters per minute.When all of the parameters have been set, load ten microliters of an extraction blank before loading the first sample. Then, separate the different lipid species using the liquid chromatography gradient, as indicated. A representative total ion chromatogram from the LC-MS data for the lipids extracted from the cells will be generated.For mass spectrometric analysis of the extracted lipids, load the samples onto a mass spectrometer equipped with a heated electro-spray ionization ion source and set the analysis parameters as outlined in the table. Use the Fourier Transform Analyzer to detect MS-1 parent ions at a resolution of 60, 000 and within the mass range of 150-2000 daltons. Then, detect the MS-2 secondary ions using the settings as indicated in the table.To identify and quantify different lipids from broad tandem LC-MS files, search LC-MS raw files containing full scan MS-1 data and data dependent MS-2 data for free, unesterified fatty acids, cardio-lipid, phytocerimide, phytosphyngocene, phosphatidylcholine, phosphatidylethanolamine, phosphatydlglycerol, phosphatydlinositol, phosphatydlserine and triacylglycerol lipid classes using a mass divided by charge tolerance of five parts per million for precursor ions and ten parts per million for product ions. Then, follow the instructions provided in the software user manual to identify internal lipid standards and lipid species with unusual fatty acid compositions. This sensitive tandem LC-MS method enables the identification and quantification of molecular species of lipids at concentrations as low as 0.165 picomolar per microliter.This limit of quantification differs from different lipid classes within a wide range of concentrations. This method can be used to increase the efficiency of ionization for lipids by using alternative mobile phase additives for the electrospray ionization mass spectrometry. Each of these alternative mobile phase additives can be used for both the normal phase and reverse phase LC columns.The collision induced dissociation method is beneficial if used in combination with the ammonium acetate mobile phase additive for the electro-spray ionization negative mode of mass spectrometry, as, under these conditions, it allows the increase in the efficiency of MS-1 lipid ion fragmentation into MS-2 products. In contrast, the high energy, collisional dissociation method is favorable if used in combination with the ammonium formate mobile phase additive for the electro-spray ionization positive mode of mass spectrometry as under these conditions, it enables an increase in the efficiency of MS-1 lipid ion fragmentation into MS-2 products. Do not mix the phases while collecting the organic phase.Close the tubes under the flow of nitrogen gas. Eliminate any bubble within the insert and connect the column in the correct direction. If the identity of any peak cannot be determined by LC-MS, MS-NMR can be used to deduce the structure of that molecule.

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Biochemistry

7.1K visualizações.  Concordia University. Este protocolo fornece um método preciso e reprodutível para quantificar dez classes lipídicas diferentes de Saccharomyces cerevisiae usando um único método de extração e um único método LC-MS. Este método permite a detecção e quantificação de espécies lipídicas isobáricas e isomericas com diferentes propriedades químicas e físicas. Para a cultura saccharomyces cerevisiae, adicione primeiro 5 ml de solução de estoque de glicose estéril de 20% para cada um dos dois frasco...