Name: quantitative metabolomics of saccharomyces cerevisiae using liquid chromatography coupled with tandem mass spectrometry
Uploaded: 2021-01-05T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Metabolomics 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, the Centre for Structural and Functional Genomics, and the Centre for Microscopy and Cellular Imaging (all 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 CRDPJ 515900 - 17). K.M. was supported by the Concordia University Armand C. Archambault Fellowship and the Concordia University Dean of Arts and Sciences Award of Excellence.

1. Making and sterilizing a medium for growing yeast
<ol>
	<li>Make 180 mL of a complete yeast extract with bactopeptone (YP) medium. The complete YP medium contains 1% (w/v) yeast extract and 2% (w/v) bactopeptone.</li>
	<li>Distribute 180 mL of the YP medium equally into four 250 mL Erlenmeyer flasks. Each of these flasks contains 45 mL of the YP medium.</li>
	<li>Sterilize the flasks with YP medium by autoclaving at 15 psi/121 &#176;C for 45 min.</li>
</ol>
2. Wild-type yeast strain
<ol>
	<...

To successfully use the protocol described here, follow the preventive measures described below. Chloroform and methanol extract various substances from laboratory plasticware. Therefore, handle them with caution. Avoid the use of plastics in steps that involve contact with any of these two organic solvents. Use borosilicate glass pipettes for these steps. Rise these pipettes with chloroform and methanol before use. Use only micropipette tips and tubes made of polypropylene that is resistant to organic solvents. During s...

Water-soluble metabolites are low-molecular-weight intermediates and products of metabolism that contribute to essential cellular processes. These evolutionarily conserved processes include the conversion of nutrients into usable energy, synthesis of macromolecules, cellular growth and signaling, cell cycle control, regulation of gene expression, stress response, post-translational regulation of metabolism, maintenance of mitochondrial functionality, vesicular cellular trafficking, autophagy, cellular aging, and regulated cell death1,2,3.
Many of these essential roles of water-soluble metabolites have been discovered by studies in the budding yeast S. cerevisiae1,3,4,7,9,14,15,16,17,18,19,20,21,22. This unicellular eukaryote is a useful model organism for dissecting mechanisms through which water-soluble metabolites contribute to cellular processes due to its amenability to advanced biochemical, genetic, and molecular biological analyses23,24,25,26. Although the LC-MS/MS methods of non-targeted metabolomics have been used for studying the roles of water-soluble metabolites in budding yeast3,18,22,27, this type of analysis requires the improvement of its versatility, robustness, sensitivity, and ability to distinguish between different structural isomers and stereoisomeric forms of these metabolites.
Recent years are marked by significant advances in applying the LC-MS/MS methods of non-targeted metabolomics to the profiling of water-soluble metabolites in vivo. However, many challenges in using this methodology remain2,28,29,30,31,32,33,34,35,36. These challenges include the following. First, the intracellular concentrations of many water-soluble metabolites are below a threshold of sensitivity for the presently used methods. Second, the efficiency of metabolic activity quenching is too low, and the extent of quenching-associated cell leakage of intracellular metabolites is too high for current methods; hence, the presently used methods under-estimate the intracellular concentrations of water-soluble metabolites. Third, the existing methods cannot differentiate the structural isomers (i.e., molecules with the same chemical formula but different atomic connectivity) or stereoisomers (i.e., molecules with the same chemical formula and atomic connectivity, but with the different atomic arrangement in space) of specific metabolites; this prevents the correct annotation of certain metabolites by the presently used methods. Fourth, the existing mass spectral online databases of parent ions (MS1) and secondary ions (MS2) are incomplete; this affects the correct identification and quantitation of specific metabolites using the raw LC-MS/MS data produced with the help of the current methods. Fifth, the existing methods cannot use a single type of metabolite extraction to recover all or most classes of water-soluble metabolites. Sixth, the existing methods cannot use a single type of the LC column to separate from each other all or most classes of water-soluble metabolites.
Here, we optimized conditions for quenching of metabolic activity within S. cerevisiae cells, maintaining most of the water-soluble metabolites within these cells before extraction, and extracting most classes of water-soluble metabolites from yeast cells. We developed a versatile, robust, and sensitive method for the LC-MS/MS-based identification and quantification of more than 370 water-soluble metabolites extracted from S. cerevisiae cells. This method of non-targeted metabolomics enables to assess the intracellular concentrations of various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The developed LC-MS/MS method permits the identification and quantification of different structural isomers and stereoisomeric forms of water-soluble metabolites with diverse structural, physical, and chemical properties.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Chemicals</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A9554</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>Bactopeptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C297-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-10</td>
			<td></td>
		</tr>
		<tr>
			<td>L-histidine</td>
			<td>Sigma</td>
			<td>H8125</td>
			<td></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>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A4564</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Thermo Scientific</td>
			<td>R37108</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Hardware equipment</td>
		</tr>
		<tr>
			<td>500 ml centrifuge bottles</td>
			<td>Beckman</td>
			<td>355664</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1100 series LC system</td>
			<td>Agilent Technologies</td>
			<td>G1312A</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter Centrifuge</td>
			<td>Beckman</td>
			<td>6254249</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter Centrifuge Rotor</td>
			<td>Beckman</td>
			<td>JA-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Centra CL2 clinical centrifuge</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital thermometer</td>
			<td>Omega</td>
			<td>HH509</td>
			<td></td>
		</tr>
		<tr>
			<td>Foam Tube Holder Kit with Retainer</td>
			<td>Thermo Scientific</td>
			<td>02-215-388</td>
			<td></td>
		</tr>
		<tr>
			<td>SeQuant ZIC-pHILIC zwitterionic-phase column (5&#181;m polymer 150 x 2.1 mm)</td>
			<td>Sigma Milipore</td>
			<td>150460</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Orbitrap Velos MS</td>
			<td>Fisher Scientific</td>
			<td>ETD-10600</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic sonicator</td>
			<td>Fisher Scientific</td>
			<td>15337416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>ZORBAX Bonus-RP, 80&#197;, 2.1 x 150 mm, 5 &#181;m</td>
			<td>Agilent Technologies</td>
			<td>883725-901</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Laboratory materials</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>Glass beads (acid-washed, 425-600 &#956;m)</td>
			<td>Sigma-Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
		<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 colspan="4">Software</td>
		</tr>
		<tr>
			<td>Compound Discoverer 3.1</td>
			<td>Fisher Scientific</td>
			<td>V3.1</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Yeast strain</td>
		</tr>
		<tr>
			<td>Yeast strain BY4742</td>
			<td>Dharmacon</td>
			<td>YSC1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative metabolomics of saccharomyces cerevisiae using liquid chromatography coupled with tandem mass spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

To improve a quantitative assessment of water-soluble metabolites within a yeast cell, we optimized the conditions of cell quenching for metabolite detection. Cell quenching for this purpose involves a rapid arrest of all enzymatic reactions within a cell31,33,37,38. Such an arrest of cellular metabolic activity is an essential step of any method for the quantit...

Watch this Scientific Journal Video about Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry at JoVE.com

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

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