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.

Representative 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). Numerous molecular species of each of these classes were identified and quantified using this LC-MS/MS method (Supplemental Table 1).

Our LC-MS/MS method was also sensitive. Indeed, it enabled the identification and quantitation of molecular species of lipids at concentrations as low as 0.165 pmol/µL (see data for phytoceramide in Table 5). This limit of quantitation differs for different lipid classes within a wide range of concentrations (Table 5).

Importantly, our method allowed identification and quantification of different isobaric or isomeric forms of lipids. Isobaric forms of lipids are lipid species with the same nominal mass (ie., sum of the masses of the most abundant isotopes) but differing exact masses³¹. Isomeric forms of lipids are lipid species with the same molecular formula but with different chemical structure³¹. For example, the use of our LC-MS/MS method distinguished between PHC (16:0_26:0) and the isobaric lipid species PC (16:0_10:0): although they have the same nominal mass value of 650, their exact masses are 650.6457 and 650.4755, respectively. Moreover, this LC-MS/MS method distinguished between two pairs of isomeric lipid species with the same molecular formula but different chemical structure: 1) PC (18:0_18:1) and PC (20:0_16:1), with molecular formula (C44H87N1O8P1) and exact mass (788.6163); and 2) PE (16:0_16:0) and PE (14:0_18:0), with the molecular formula (C37H75N1O8P1) and exact mass (692.5224).

Our LC-MS/MS method can be used to increase the efficiency of ionization for lipids of all classes, thus improving the identification and quantitation of major cellular lipids. Such improvement can be achieved by using alternative mobile phase additives for the ESI MS (Table 6). These alternative phases include ammonium formate, ammonium formate with formic acid, ammonium acetate, ammonium acetate with acetic acid, and ammonium acetate with formic acid. Each of these alternative mobile phase additives can be used for both the normal-phase and reverse-phase LC columns (Table 6).

Another advantage of our LC-MS/MS method consists in the ability to use two different methods for the fragmentation of precursor ions (MS1) of lipids into MS2 products. These two methods are high-energy collisional dissociation (HCD) and collision-induced dissociation (CID)³². We found that the CID method is beneficial if used in combination with the ammonium acetate mobile phase additive for the ESI (-) mode of MS, as under these conditions it allows an increase in the efficiency of MS1 lipid ion fragmentation into MS2 products for PHC, CL, FFA, PE, PG, PI, and PS (Table 7). In contrast, the HCD method is favorable if used in combination with the ammonium formate mobile phase additive for the ESI (+) mode of MS, as under these conditions it enables an increase in the efficiency of MS1 lipid ion fragmentation into MS2 products for PC, PHS and TAG (Table 8).

Figure 1: The total ion chromatogram (TIC) from liquid chromatography/mass spectrometry (LC/MS) data of lipids that were extracted from cells of the wild type strain BY4742. The TIC of lipids separated by LC on a reverse-phase column CSH C18 and detected by MS of parent ions that were created using the negative electrospray ionization mode. Please click here to view a larger version of this figure.

Figure 2: A chromatogram of two isomeric phosphatidylserine forms (34:0) that have the same m/z value (M-H) of 762.5294 but different retention times of 7.65 min and 8.49 min. The lipids were extracted from cells of the wild type strain BY4742 and separated by liquid chromatography on a reverse-phase column CSH C18. Please click here to view a larger version of this figure.

Figure 3: MS1 spectra of two isomeric phosphatidylserine species (34:0) that have the same m/z value (M-H) of 762.5294 but different retention times of 7.65 min and 8.49 min. The lipids were extracted from cells of the wild type strain BY4742, separated by liquid chromatography on a reverse-phase column CSH C18 (as shown in Figure 2) and detected by mass spectrometry of parent (MS1) ions that were created using the negative electrospray ionization mode. (A) The MS1 spectrum of a phosphatidylserine form (34:0) with the m/z value (M-H) of 762.5294 and the retention time of 7.65 min. (B) The MS1 spectrum of a phosphatidylserine form (34:0) with the m/z value (M-H) of 762.5294 and the retention time of 8.49 min. Please click here to view a larger version of this figure.

Figure 4: MS2 spectra of two isomeric phosphatidylserine species (34:0) that have the same m/z value (M-H) of 762.5294 but different retention times of 7.65 min and 8.49 min. The lipids were extracted from cells of the wild type strain BY4742, separated by liquid chromatography on a reverse-phase column CSH C18 (as shown in Figure 2) and detected by mass spectrometry of MS1 ions (as shown in Figure 3). Secondary ions (MS2) were then detected by mass spectrometry. (A) The MS2 spectrum of a phosphatidylserine form (34:0) with the m/z value (M-H) of 762.5294 and the retention time of 7.65 min. The loss of a serine moiety produced an ion with the m/z value (M-H) of 675.6149. (B) The MS2 spectrum of a phosphatidylserine form (34:0) with the m/z value (M-H) of 762.5294 and the retention time of 8.49 min. The loss of a serine moiety produced an ion with the m/z value (M-H) of 675.8843. Please click here to view a larger version of this figure.

Detection mode	Lipid class	Hydrophobic tail composition	Calculated m/z value	Concentration (pmoles/µl)
Negative	Ceramide	18:1_17:0	550.5204675	226
Negative	Cardiolipin	14:0_14:0_14:0_14:0	1239.839755	196
Negative	Free fatty acid	19:0	297.2799035	837
Negative	Phosphatidylethanolamine	15:0_15:0	662.4766305	377
Negative	Phosphatidylglycerol	15:0_15:0	693.4712115	349
Negative	Phosphatidylinositol	17:0_20:4	871.5342065	3
Negative	Phosphatidylserine	17:0_17:0	762.5290605	318
Positive	Phosphatidylcholine	13:0_13:0	650.4755335	385
Positive	Phytosphingosine	16:1	272.2584055	225
Positive	Triacylglycerol	28:1_10:1_10:1	818.7232155	367

Table 1: The composition of a mixture of internal lipid standards. Internal lipid standards were prepared in chloroform/methanol (2:1) mixture. Detection mode refers to a positive or negative mode of parent ions detection using an Orbitrap Velos Mass Spectrometer equipped with electrospray ionization (ESI) ion source. The calculated m/z values are for the adducts of lipids.

FTMS + p resolution	60000
Mass range (dalton)	150-2000
Ion source type	HESI
Capillary temperature (°C)	300
Source heater temperature (°C)	300
Sheath gas flow	10
Aux gas flow	5
Positive polarity voltage (kV)	3
Negative polarity voltage (kV)	3
Source current (µA)	100

Table 2: The Orbitrap Velos Mass Spectrometer's settings used to analyze lipids that were separated by LC. Abbreviations: FTMS + p = Fourier transform-based mass spectrometry in the ESI (+) mode; HESI = heated electrospray ionization.

Instrument polarity	Positive	Negative
Activation type	High-energy-induced-collision-dissociation	Collision-induced-dissociation
Minimal signal required	5000	5000
Isolation width	2	2
Normalized collision energy	55	35
Default charge state	2	2
Activation time	0.1	10
FTMS + C resolution	7500
5 most intense peaks were selected for ms/ms

Table 3: The Orbitrap Velos Mass Spectrometer's settings used to detect secondary ions (MS2). Abbreviation: FTMS + C = Fourier transform-based mass spectrometry in the ESI (+) mode.

Identification
Database	Orbitrap
Peak detection	Recall isotope (ON)
Search option	Product search Orbitrap
Search type	Product
Experiment type	LC-MS
Precursor tolerance	10 ppm
Product tolerance	High-energy-induced-collision-dissociation [ESI (+) mode]: 20 ppm
	Collision-induced-dissociation [ESI (-) mode]: 0.5 Daltons
Quantitation
Execute quantitation	ON
m/z tolerance	-5.0; +5.0
Tolerance type	ppm
Filter
Top rank filter	ON
Main node filter	Main isomer peak
m-score threshold	5
c-score threshold	2
FFA priority	ON
ID quality filter	A: Lipid class & all fatty acids are completely identified
	B: Lipid class & some fatty acids are identified
	C: Lipid class or FA are identified
	D: Lipid identified by other fragment ions (H2O, etc.)
Lipid Class
High-energy-induced-collision-dissociation [ESI (+) mode]	PC, TAG
Collision-induced-dissociation [ESI (-) mode]	CER, CL, FFA, PE, PG, PI, PS
Ions
High-energy-induced-collision-dissociation [ESI (+) mode]	+ H; + NH4; + Na
Collision-induced-dissociation [ESI (-) mode]	- H; - 2H; - HCOO

Table 4: The search parameters used to identify different lipid classes and species with the help of the Lipid Identification software "Lipid Search" (V 4.1). Abbreviations: CER = ceramide; CL = cardiolipin; FFA = free (unesterified) fatty acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PG = phosphatidylglycerol; PI = phosphatidylinositol; PS = phosphatidylserine; TAG = triacylglycerol.

Table 5: The lowest concentrations of molecular species of different lipid classes that can be identified and quantitated with the help of our LC-MS/MS method. An estimate of the lowest quantifiable concentration for each lipid class is based on the MS peak areas for its internal standard (these MS peak areas and lipid standard concentrations are displayed in bold) and its representative molecular form present at the lowest detectable concentration. Data are presented as mean values of two independent experiments, for each of which three technical replicates were performed. Please click here to view this table (Right click to download).

Lipid standard	ESI mode	AmF	AmF/FA	AmAc	AmAc/AA	AmAc/FA
PHC	-	74	75.2	85.8	77	74.8
CL	-	72.9	75.1	78.3	68.4	74
FFA	-	77.1	77.2	75.5	84	72.9
PE	-	98	96	95	91.5	86
PG	-	64	45.1	75.9	60.5	55
PI	-	79.3	76	82.5	80.3	77
PS	-	73.7	61.6	85.8	81.4	73.7
PC	+	93.5	86.9	69.3	60.5	62.7
PHS	+	89.1	79.2	78.1	73.7	69.3
TAG	+	92.4	88	86.9	80.3	84.7

Table 6: The effects of alternative mobile phase additives for the ESI MS on the efficiencies of ionization for lipids of different classes. Different lipids were separated from each other by reverse-phase liquid chromatography. Commercial lipid standards that belong to different classes of lipids are named in Table 1. The ESI (-) or ESI (+) mode of ionization was used for MS in the presence of different mobile phase additives. The percentage of ionization for each lipid standard is shown as a mean value from three technical replicates. For each lipid, the ionization percentage was calculated based on the MS peak area. A value of the highest ionization efficiency for each lipid is displayed in bold. Abbreviations: AmF = ammonium formate; AmF/FA = ammonium formate with formic acid; AmAc = ammonium acetate; AmAc/AA = ammonium acetate with acetic acid; AmAc/FA = ammonium acetate with formic acid; CL = cardiolipin; FFA = free (unesterified) fatty acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PG = phosphatidylglycerol; PHC = phytoceramide; PHS = phytosphingosine; PI = phosphatidylinositol; PS = phosphatidylserine; TAG = triacylglycerol.

Lipid standard	ESI mode	AmF	AmAc
PHC	-	75	83.8
CL	-	70.9	79.3
FFA	-	76.1	74.5
PE	-	98	95
PG	-	64	75.9
PI	-	79.3	82.5
PS	-	71.5	84.8
PC	+	52.3	60.5
PHS	+	78.4	75.2
TAG	+	65.7	69.7

Table 7: The effect of the collision-induced dissociation (CID) method on the efficiency of precursor ions (MS1) fragmentation. Commercial lipid standards that belong to different classes of lipids are named in Table 1. The ESI (-) or ESI (+) mode of ionization was used for MS, in the presence of an ammonium formate (AmF) or ammonium acetate (AmAc) mobile phase additive. The percentage of MS1 lipid ions that were fragmented into MS2 products is shown as a mean value from three technical replicates. For each lipid, the ionization percentage was calculated based on the MS2 peak area. A value of the highest percentage of MS1 lipid ions that were fragmented is displayed in bold for each lipid (compare with the data presented in Table 8). This value is the highest if the efficiency of MS2 product ion formation is the highest. Abbreviations: CL = cardiolipin; FFA = free (unesterified) fatty acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PG = phosphatidylglycerol; PHC = phytoceramide; PHS = phytosphingosine; PI = phosphatidylinositol; PS = phosphatidylserine; TAG = triacylglycerol.

Lipid standard	ESI mode	AmF	AmAc
PHC	-	68.4	65.4
CL	-	74.3	75.2
FFA	-	84.2	81.2
PE	-	85.1	73.1
PG	-	68.4	67.1
PI	-	58.7	55.8
PS	-	67.4	68.5
PC	+	92.5	65.3
PHS	+	87.1	75.1
TAG	+	91.4	84.9

Table 8: The effect of the high-energy collisional dissociation (HCD) method on the efficiency of precursor ions (MS1) fragmentation. Commercial lipid standards that belong to different classes of lipids are named in Table 1. The ESI (-) or ESI (+) mode of ionization was used for MS, in the presence of an ammonium formate (AmF) or ammonium acetate (AmAc) mobile phase additive. The percentage of MS1 lipid ions that were fragmented into MS2 products is shown as a mean value from three technical replicates. For each lipid, the ionization percentage was calculated based on the MS2 peak area. A value of the highest percentage of MS1 lipid ions that were fragmented is displayed in bold for each lipid (compare with the data presented in Table 7). This value is the highest if the efficiency of MS2 product ion formation is the highest. Other abbreviations: CL = cardiolipin; FFA = free (unesterified) fatty acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PG = phosphatidylglycerol; PHC = phytoceramide; PHS = phytosphingosine; PI = phosphatidylinositol; PS = phosphatidylserine; TAG = triacylglycerol.

Supplemental Table 1: A list of molecular species of 10 different lipid classes identified and quantified in S. cerevisiae cells with the help of our LC-MS/MS method. These lipid classes included free (unesterified) fatty acids (FFA), cardiolipin (CL), phytoceramide (PHC), phytosphingosine (PHS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TAG). S. cerevisiae cells were cultured in the synthetic minimal YNB medium initially containing 2% glucose. Aliquots of yeast cells for lipid extraction and LC-MS/MS analysis of extracted lipids were recovered on days 1, 2, 3, 4, 6, 8, and 10 of culturing. All lipid species of each lipid class that were identified in yeast cells recovered on different days of culturing are shown. Some of these lipid species were present only in chronologically young yeast cells recovered on days 1-4 of culturing, others only in chronologically old yeast cells recovered on days 6-10 of culturing, whereas some were present in both chronologically young and old yeast cells. The highest MS peak area for each lipid species of each lipid class identified on a certain day of culturing is shown. Lipid standards of different lipid classes that were used for quantitation of other species within this lipid class are displayed in red color. The calculated m/z values are for the adducts of lipids. Abbreviation: ESI (-) = the ESI (-) mode of MS; ESI (+) = the ESI (+) mode of MS. Data are presented as mean values of two independent experiments, for each of which three technical replicates were performed. Please click here to view this table (Right click to download).

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 methanol before use.

2. During lipid extraction by methanol/chloroform (17:1) mixture, use a borosilicate glass pipette to transfer the lower organic phase (~400 µL) to a 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.

3. During sample preparation for LC-MS/MS, it is important to eliminate all air bubbles in the glass vials before inserting the vials into a wellplate.

4. To achieve a complete solubilization of ammonium formate and ammonium acetate in mobile phases A and B prior to lipid separation by LC, dissolve each salt in 500 µL of nano-pure water before mixing the solution with the mobile phase and sonicating for 20 min.

5. Do not store the lipid film formed after solvent evaporation for a long period of time prior to running. We store this film at -80 °C for no more than a week before dissolving it in the acetonitrile/2-propanol/nano-pure water (65:35:5) mixture and then subjecting the lipids to LC-MS/MS.

The LC-MS/MS method described here is a versatile, robust, and sensitive technique for a quantitative assessment of many lipid species comprising the cellular lipidome of yeast or any other eukaryotic organism. The method enables the identification and quantification of different isobaric or isomeric lipid species, allows to use alternative mobile phase additives for the ESI MS to promote lipid ionization and to make lipid identification and quantification more efficient, and can use both HCD and CID methods for the fragmentation or activation of MS1 lipid ions.

We use this LC-MS/MS method to study age-related changes in the cellular and organellar lipidomes during chronological aging of the budding yeast S. cerevisiae. We also employ this method to investigate how many aging-delaying genetic, dietary, and pharmacological interventions influence lipid composition of the entire S. cerevisiae cell and its various organelles. Because of its versatility, robustness, and sensitivity, this LC-MS/MS method can be successfully used for the quantitative assessment of the cellular and organellar lipidomes in eukaryotic organisms across phyla.

Materials

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