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

Summary

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.

Abstract

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/µL. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Introduction

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 death^1,2,3.

Many of t....

Protocol

1. Making and sterilizing a medium for growing yeast

1. 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.
2. 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.
3. Sterilize the flasks with YP medium by autoclaving at 15 psi/121 °C for 45 min.

2. Wild-type yeast strain

1. Use the BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) strain.

3. Growing yeast in th....

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Chemicals
Acetonitrile	Fisher Scientific	A9554
Ammonium acetate	Fisher Scientific	A11450
Ammonium bicarbonate	Sigma	9830
Bactopeptone	Fisher Scientific	BP1420-2
Chloroform	Fisher Scientific	C297-4
Glucose	Fisher Scientific	D16-10
L-histidine	Sigma	H8125
L-leucine	Sigma	L8912
L-lysine	Sigma	L5501
Methanol	Fisher Scientific	A4564
Methanol	Fisher Scientific	A4564
Propidium iodide	Thermo Scientific	R37108
Uracil	Sigma	U0750
Yeast extract	Fisher Scientific	BP1422-2
Hardware equipment
500 ml centrifuge bottles	Beckman	355664
Agilent 1100 series LC system	Agilent Technologies	G1312A
Beckman Coulter Centrifuge	Beckman	6254249
Beckman Coulter Centrifuge Rotor	Beckman	JA-10
Centra CL2 clinical centrifuge	Thermo Scientific	004260F
Digital thermometer	Omega	HH509
Foam Tube Holder Kit with Retainer	Thermo Scientific	02-215-388
SeQuant ZIC-pHILIC zwitterionic-phase column (5µm polymer 150 x 2.1 mm)	Sigma Milipore	150460
Thermo Orbitrap Velos MS	Fisher Scientific	ETD-10600
Ultrasonic sonicator	Fisher Scientific	15337416
Vortex	Fisher Scientific	2215365
ZORBAX Bonus-RP, 80Å, 2.1 x 150 mm, 5 µm	Agilent Technologies	883725-901
Laboratory materials
2-mL Glass sample vials with Teflon lined caps	Fisher Scientific	60180A-SV9-1P
Glass beads (acid-washed, 425-600 μm)	Sigma-Aldrich	G8772
Hemacytometer	Fisher Scientific	267110
15-mL High-speed glass centrifuge tubes with Teflon lined caps	PYREX	05-550
Software
Compound Discoverer 3.1	Fisher Scientific	V3.1
Yeast strain
Yeast strain BY4742	Dharmacon	YSC1049