Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Summary

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.

Abstract

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/µL. 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.

Introduction

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 death^{1,2,3,4,5,6,7,8,9,10}.

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 processes^{4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20}. 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 analyses^{21,22,23,24,25}. 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 biology^{1,2,3,26,27,28,29,30}.

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.

Protocol

1. Preparation of sterile media for culturing yeast

1. Prepare 90 mL of a complete YP medium that contains 1% (w/v) yeast extract and 2% (w/v) bactopeptone.
2. 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.
3. Divide 90 mL of the complete YP medium equally into two 250 mL Erlenmeyer flasks (i.e., 45 mL each).
4. Divide 90 mL of the synthetic minimal YNB medium into two 250 mL Erlenmeyer flasks (i.e., 45 mL each).
5. Autoclave the flasks with YP and YNB media at 15 psi/121 °C for 45 min prior to use.

2. Yeast strain

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

3. Culturing yeast in the complete YP medium with glucose

1. Autoclave a 20% (w/v) stock solution of glucose at 15 psi/121 °C for 45 min prior to use.
2. Add 5 mL of the sterile 20% (w/v) stock solution of glucose to each of the two Erlenmeyer flasks containing the sterile YP medium for a final concentration of 2% glucose (w/v).
3. Use a microbiological loop to inoculate cells of the wild type strain BY4742 into each of the two Erlenmeyer flasks containing the YP medium with glucose.
4. Culture the cells overnight at 30 °C with rotational shaking at 200 rpm.
5. Take an aliquot of yeast culture. Use a hemocytometer to determine the total number of yeast cells per mL of culture.

4. Transferring yeast to and culturing them in the synthetic minimal YNB medium with glucose

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

5. Preparation of reagents, labware and equipment for lipid extraction

1. Prepare the following: 1) high grade (>99.9%) chloroform; 2) high grade (>99.9%) methanol; 3) 28% (v/v) ammonium hydroxide solution in nano-pure water; 4) glass beads (acid-washed, 425-600 µM); 5) a vortex with appropriate adapter; 6) 15 mL high-speed glass centrifuge tubes with polytetrafluoroethylene lined caps; 7) 17:1 and 2:1 mixtures of chloroform and methanol; 8) a chloroform/methanol (2:1) mixture with 0.1% ammonium hydroxide (v/v); 9) ABC solution (155 mM ammonium bicarbonate, pH = 8.0); 10) a mixture of internal lipid standards prepared in a 2:1 mixture of chloroform and methanol as indicated in Table 1; and 11) 2 mL glass sample vials with polytetrafluoroethylene lined caps for the extraction of cellular lipids.

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

1. Prepare the following: 1) acetonitrile/2-propanol/nano-pure water (65:35:5) mixture; 2) a vortex with appropriate adapter; 3) an ultrasonic sonicator; 4) glass vials with inserts for a wellplate; 5) an LC system equipped with a binary pump, degasser, and an autosampler; 6) a C18 reverse-phase column (2.1 mm; 75 mm; pore size 130 Å; pH range of 1-11) coupled to a pre-column system; 7) mixture A: acetonitrile/water (60:40); and 8) mixture B: isopropanol/acetonitrile (90:10).

7. Lipid extraction from yeast cells

1. Take an aliquot of yeast culture. Use a hemocytometer or measure OD₆₀₀ to determine the total number of yeast cells per mL of culture.
2. Take a volume of yeast culture that contains a total number of 5.0 x 10⁷ cells (3.3 units OD₆₀₀). Place this volume of culture into a prechilled 1.5 mL microcentrifuge tube.
3. Harvest the cells by centrifugation at 16,000 x g for 1 min at 4 °C. Discard the supernatant.
4. Add 1.5 mL of ice-cold nano-pure water and wash the cells by centrifugation at 16,000 x g for 1 min at 4 °C. Discard the supernatant.
5. Add 1.5 mL of ice-cold ABC solution and wash the cells by centrifugation at 16,000 x g for 1 min at 4 °C. Discard the supernatant. The cell pellet can be stored at -80 °C prior to lipid extraction.
6. To begin the lipid extraction, thaw the cell pellet on ice.
7. Resuspend the cell pellet in 200 µL of ice-cold nano-pure water. Transfer the cell suspension to a 15 mL high-strength glass screw top centrifuge tube with a polytetrafluoroethylene lined cap. Add the following to this tube: 1) 25 µL of the mixture of internal lipid standards prepared in chloroform/methanol (2:1) mixture; 2) 100 µL of 425-600 µM acid-washed glass beads; and 3) 600 µL of chloroform/methanol (17:1) mixture.
8. Vortex the tube at high speed for 5 min at room temperature (RT) to disrupt the cells.
9. Vortex the tube at low speed for 1 h at RT to facilitate the extraction of lipids.
10. Incubate the sample for 15 min on ice to promote protein precipitation and the separation of the aqueous and organic phases from each other.
11. Centrifuge the tube in a clinical centrifuge at 3,000 x g for 5 min at RT. This centrifugation step allows to separate the upper aqueous phase from the lower organic phase, which contains all lipid classes.
12. Use a borosilicate glass pipette to transfer the lower organic phase (~400 µL) to another 15 mL high-strength glass screw top centrifuge tube with a polytetrafluoroethylene lined cap. Do not disrupt the glass beads or upper aqueous phase during such transfer. Keep the lower organic phase under the flow of nitrogen gas.
13. Add 300 µL of chloroform-methanol (2:1) mixture to the remaining upper aqueous phase to allow the extraction of sphingolipids and PA, PS, PI, and CL. Vortex the tube vigorously for 5 min at RT.
14. Centrifuge the tube in a clinical centrifuge at 3,000 x g for 5 min at RT.
15. Use a borosilicate glass pipette to transfer the lower organic phase (~ 200 µL) formed after centrifugation to the organic phase collected at step 7.13.
16. Use the flow of nitrogen gas to evaporate the solvent in the combined organic phases. Close the tubes containing the lipid film under the flow of nitrogen gas. Store these tubes at -80 °C.

8. Separation of extracted lipids by LC

1. Add 500 µL of acetonitrile/2-propanol/nano-pure water (65:35:5) mixture to a tube containing the lipid film obtained at step 7.16. Vortex the tube 3x for 10 s at RT.
2. Subject the content of the tube to ultrasonic sonication for 15 min. Vortex the tube 3x for 10 s at RT.
3. Take 100 µL of a sample from the tube and add it to a glass vial with an insert used for a wellplate. Eliminate air bubbles in the insert before pacing it into the wellplate.
4. Use an LC system to separate different lipid species on a reverse-phase C18 column CSH coupled to a pre-column system (see Table of Materials). During the separation, maintain the column at 55 °C and at a flow rate of 0.3 mL/min. Keep the sample in the wellplate at RT.
5. Use the mobile phases that consist of mixture A (acetonitrile/water [60:40 (v/v)]) and mixture B (isopropanol/acetonitrile [90:10 (v/v)]). For a positive mode of the detection of parent ions created using the electrospray ionization (ESI) ion source, the ESI (+) mode, the mobile phases A and B contain ammonium formate at the final concentration of 10 mM. For a negative mode of parent ions detection, the ESI (-) mode, the mobile phases A and B contain ammonium acetate at the final concentration of 10 mM.
6. Use a sample volume of 10 µL for the injection into both the ESI (+) and ESI (-) mode.
7. Separate different lipid species by LC using the following LC gradient: 0-1 min 10% (phase B); 1-4 min 60% (phase B); 4-10 min 68% (phase B); 10-21 97% (phase B); 21-24 min 97% (phase B); 24-33 min 10% (phase B).
8. Run extraction blanks as the first sample, between every four samples, and as the last sample. Subtract the background to normalize data.
9. A representative total ion chromatogram from LC/MS data of lipids extracted from cells of the wild type strain BY4742 is shown in Figure 1.

9. Mass spectrometric analysis of lipids separated by LC

1. Use a mass spectrometer equipped with a HESI (heated electrospray ionization) ion source to analyze lipids that were separated by LC. Use the settings provided in Table 2.
2. Use the Fourier transform analyzer to detect parent ions (MS1) at a resolution of 60,000 and within the mass range of 150-2,000 Da.
3. Use the settings provided in Table 3 to detect secondary ions (MS2).

10. Identification and quantitation of different lipid classes and species by processing of raw data from LC-MS/MS

1. See the Table of Materials for software to carry out the identification and quantitation of different lipids from raw LC-MS/MS files. This software uses the largest lipid database, containing more than 1.5 million lipid ion precursors (MS1) and their predicted fragment ions (MS2). The software also uses MS1 peaks for lipid quantitation and MS2 for lipid identification. A representative chromatogram of two isomeric phosphatidylserine forms (34:0) that have the same m/z value but different retention times, as well as their MS1 and MS2 spectra, are shown in Figure 2, Figure 3, and Figure 4, respectively.
2. Search LC-MS raw files containing full-scan MS1 data and data-dependent MS2 data for free (unesterified) fatty acids (FFA), cardiolipin (CL), phytoceramide (PHC), phytosphingosine (PHS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TAG) lipid classes using an m/z tolerance of 5 ppm for precursor ions and 10 ppm for product ions. Other search parameters are shown in Table 4. Follow the instructions provided in the software user manual. The identities of internal lipid standards and lipid species with unusual fatty acid composition need to be verified manually.
3. To identify and quantitate different lipid classes and species with the help of freely available open-source alternatives for the Lipid Search software, use the Lipid Data Analyzer (http://genome.tugraz.at/lda2/lda_download.shtml), MZmine 2 (http://mzmine.github.io/), or XCMS (https://bioconductor.org/packages/release/bioc/html/xcms.html) software to process raw data from LC-MS/MS.

Results

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

Discussion

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 ...

Materials

List of materials used in this article

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