Name: quantitative analysis of the cellular lipidome of saccharomyces cerevisiae using liquid chromatography coupled with tandem mass spectrometry
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Description: Watch this Scientific Journal Video about Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry at JoVE.com

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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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. 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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 ...

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Biochemistry

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