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 these essential roles of water-soluble metabolites have been discovered by studies in the budding yeast S. cerevisiae^{1,3,4,7,9,14,15,16,17,18,19,20,21,22}. 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 analyses^23,24,25,26. Although the LC-MS/MS methods of non-targeted metabolomics have been used for studying the roles of water-soluble metabolites in budding yeast^3,18,22,27, 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 remain^{2,28,29,30,31,32,33,34,35,36}. 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.

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 the YP medium containing 2% glucose

1. Sterilize a 20% (w/v) stock solution of glucose by autoclaving at 15 psi/121 °C for 45 min.
2. Add 5 mL of the autoclaved 20% (w/v) stock solution of glucose to each of the two Erlenmeyer flasks with 45 mL of the sterilized YP medium. The final concentration glucose in the YP medium is 2% (w/v).
3. Use a microbiological loop to inoculate yeast cells into each of the two Erlenmeyer flasks with the YP medium containing 2% glucose.
4. Grow the yeast cells overnight at 30 °C in a rotational shaker set at 200 rpm.
5. Take an aliquot of yeast culture. Determine the total number of yeast cells per mL of culture. Count cells using a hemocytometer.

4. Cell transfer to and cell grows in the YP medium with 2% glucose

1. Add 5 mL of the sterilized 20% (w/v) stock solution of glucose to each of the remaining two Erlenmeyer flasks with the autoclaved YP medium. The final concentration of glucose is 2% (w/v).
2. Transfer a volume of the overnight yeast culture in YP medium with 2% glucose that contains the total number of 5.0 x 10⁷ cells into each of the two Erlenmeyer flasks with the YP medium containing 2% glucose. Use a sterile pipette for the cell transfer.
3. Grow the yeast cells for at least 24 h (or more, if the experiment requires) at 30 °C in a rotational shaker set at 200 rpm.

5. Making reagents, preparing labware, and setting up equipment for cell quenching

1. Prepare the following: 1) a quenching solution (60% high-grade (>99.9%) methanol in 155 mM ammonium bicarbonate (ABC) buffer, pH = 8.0); 2) an ice-cold ABC solution (pH = 8.0); 3) a digital thermometer capable of measuring up to -20 °C; 4) 500 mL large centrifuge bottles; 5) a pre-cooled high-speed centrifuge with a pre-cooled rotor and pre-cooled 500 mL centrifuge bottles for this rotor, all at -5 °C; 6) metabolite extraction tubes (15 mL high-speed glass centrifuge tubes with polytetrafluoroethylene lined caps); and 7) dry ice.

6. Cell quenching

1. Use a hemocytometer to determine the number of yeast cells per mL of YP with 2% glucose culture.
2. Transfer a volume of the culture in YP medium with 2% glucose that contains the total number of 5.0 x 10⁸ cells into pre-cooled 500 mL centrifuge bottles.
3. Quickly fill the centrifuge bottle containing the cells up to the volume of 200 mL with a quenching solution stored at -20 °C.
4. Centrifuge the bottles in a high-speed centrifuge at 11,325 x g for 3 min at -5 °C.
5. Quickly and tenderly recover the bottle from the centrifuge; gently unscrew the lid and remove the supernatant without disturbing the pellet.
6. Quickly resuspend the cell pellet in 10 mL of ice-cold ABC buffer and transfer the suspension into a 15 mL high-speed glass centrifuge tube with a polytetrafluoroethylene-lined cap for metabolite extraction.
7. Collect cells by centrifugation in a clinical centrifuge at 3,000 x g for 3 min at 0 °C.
8. Quickly remove the supernatant and place the tube on dry ice to begin metabolite extraction or store the tube at -80 °C until extraction.

7. Preparation of reagents, labware and equipment for metabolite extraction

1. Prepare the following: 1) LC-MS grade chloroform; 2) LC-MS grade methanol; 3) LC grade nano-pure water; 4) LC-MS grade (ACN); 5) glass beads (acid-washed, 425-600 µm); 6) a vortex with a foam tube holder kit with retainer; 7) 15 mL high-speed glass centrifuge tubes with polytetrafluoroethylene-lined caps; 8) MS vials; 9) dry ice; and 10) 1.5 mL tubes washed once with ethanol, once with ACN and once with nano-pure water.
   ​NOTE: Use only micropipette tips and tubes made of polypropylene that is resistant to organic solvents.

8. Metabolite extraction

1. To the metabolites kept on dry ice or stored at -80 °C tube from step 6.7, add the following: 1) 2 mL of chloroform stored at -20 °C; 2) 1 mL of methanol stored at -20 °C; 3) 1 mL of ice-cold nano-pure water; and 4) 200 µL of 425-600 µm acid-washed glass beads.
2. Cover and close the mouth of a tube with aluminum foil. Place tubes in a foam tube holder kit with retainer and vortex them for 30 min at medium speed (i.e., at a speed that is set at 6, with 12 being the maximum speed of a vortex) at 4 °C to facilitate metabolite extraction.
3. Incubate the tube for 15 min on ice (NOT dry ice!) to promote protein precipitation and the separation of the upper aqueous from the lower organic phase.
4. Centrifuge the tube in a clinical centrifuge at 3,000 x g for 10 min at 4 °C. This centrifugation step allows separating the upper aqueous phase (which contains water-soluble metabolites) from the middle layer (which contains cell debris and proteins) and from the lower organic phase (which contains mostly lipids).
5. Use a micropipette to transfer the upper aqueous phase (400 µL) to a washed and labeled 1.5 mL tube containing 800 µL of ACN that was stored at -20 °C.
   NOTE: There will be white cloud precipitation after adding the upper aqueous phase to ACN kept at -20 °C.
6. Centrifuge the tube with the sample in a tabletop centrifuge at 13,400 x g for 10 min at 4 °C.
   ​NOTE: The white cloud precipitation will disappear after centrifugation.
7. Transfer 800 µL from the upper portion of a liquid in the tube to a labeled MS vial. Store the sample at 0 °C until it is analyzed by LC-MS/MS.

9. Preparation of reagents, labware, and equipment for LC

1. Prepare the following: 1) a vortex; 2) an ultrasonic sonicator; 3) MS glass vials; 4) an LC system equipped with a binary pump, degasser, and autosampler; 5) a zwitterionic-phase chromatography column (5 µm polymer, 150 x 2.1 mm) named in Table of Materials; 6) a column heater; and 7) mobile phases, including phase A (5:95 ACN:water (v/v) with 20 mM ammonium acetate, pH = 8.0) and phase B (100% ACN).

10. Separation of extracted metabolites by LC

1. Subject the content of the MS vial to ultrasonic sonication for 15 min.
2. Vortex the MS vial 3x for 10 sec at room temperature (RT).
3. Place the MS vial into the well plate.
4. During chromatograph, maintain the column at 45 °C and a flow rate of 0.250 mL/min. Keep the sample in the well plate at 0 °C. Refer to Table 1 for the LC gradients that need to be used during chromatography.
   NOTE: A representative total ion chromatogram of water-soluble metabolites that were extracted from cells of the wild-type strain BY4742 is shown in Figure 1. The metabolites separated by LC were identified and quantified by mass spectrometric analysis that was performed in positive ionization [ESI (+)] mode, as described for step 11.

11. Mass spectrometric analysis of metabolites separated by LC

1. Use a mass spectrometer equipped with heated electrospray ionization (HESI) for the identification and quantitation of water-soluble metabolites that were separated by LC. Use the mass spectrometer's analyzer for MS1 ions and the mass spectrometer's detector for MS2 ions. Use the settings provided in Table 2 and Table 3 for the data-dependent acquisition of MS1 and MS2 ions, respectively.
2. Use a sample volume of 10 µL for the injection in both the ESI (+) and ESI (-) modes.

12. Identification and quantitation of different metabolites by the processing of raw data from LC-MS/MS

1. Use the software named in Table of Materials to conduct the identification and quantitation of different water-soluble metabolites from raw LC-MS/MS files. This software uses MS1 for metabolite quantitation and MS2 for metabolite identification. The software exploits the most extensively curated mass spectral fragmentation library to annotate the metabolites using the LC-MS/MS raw data by matching MS spectra. This software also uses the exact mass of MS1 and isotope pattern match to annotate metabolites using online databases. See Figure 2 for details.
2. Use the library of databases and spectra, which is freely available online (https://www.mzcloud.org), to search for MS2 spectra of the raw data.

13. Membrane integrity assay by propidium Iodide (PI) staining and fluorescence microscopy

1. After cell quenching performed as described for step 6, wash the quenched cells thoroughly with 15 mL of ABC buffer to remove the quenching solution. Collect cells by centrifugation at 3,000 x g for 5 min at 0 °C.
2. Resuspend the cell pellet in 1 mL of ABC buffer and add 0.5 mL of the PI solution (0.5 mg/mL).
3. Vortex a tube with the sample 3x for 10 s and incubate it for 10 min in the dark and on ice.
4. Centrifuge the tube with the sample in a tabletop centrifuge at 13,400 x g for 10 min at 4 °C.
5. Remove the supernatant and resuspend the pellet in 1 mL of ABC buffer.
6. Centrifuge the tube at 13,400 x g for 10 min at 4 °C and remove the supernatant. Repeat this step 2 more times to remove the PI bound to the cell surface.
7. Resuspend the pellet in 300 µL of ABC buffer. Place 10 µL of the suspension on the surface of a microscope slide.
8. Capture the differential interference contrast (DIC) and fluorescence microscopy images with a fluorescence microscope. Use a filter set up at the excitation and emission wavelengths of 593 nm and 636 nm (respectively).
9. Use a software to count the total cell number (in the DIC mode) and the number of fluorescently stained cells. Also, use this software to determine the intensity of staining for individual cells.

Results

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 cell^31,33,37,38. Such an arrest of cellular metabolic activity is an essential step of any method for the quantit...

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