Pulse-labeling and mass-tagging lipids
1. Prepare precultures of the relevant yeast strains in 25ml SD medium such that the optical density at 600 nm ([email protected]) is between 1 and 2 the next day morning.
2. Measure OD and collect 8 ODs in 15 ml falcon tubes. Snap-freeze the pellet. This will be the first time point (t0)
3. Dilute the remainder of the preculture in SD medium to 0.8 OD600/ml in 25 ml.
4. Freshly prepare a 1 M stock of deuterated methionine in SD medium. Decide the volume of the stock solution based on the number of yeast strains.
5. Initiate pulse-labeling by adding 2mM deuterated methionine to the diluted precultures.
6. At different time points (in our case, t3.5, 6.5, 10.5, 14.5 hours), pellet 8 OD cells, snap-freeze and store at -80oC.
Lipids were extracted as described previously with minor modifications1.
1. Thaw the frozen cells on ice for 5 min.
2. Resuspend cells in 1 ml ice-cold water and transfer them to glass tubes.
3. Pellet the cells and remove water.
4. Add 300µL glass beads and 1.5 ml of extraction solvent containing ethanol, water, diethyl ether, pyridine, and 4.2 N ammonium hydroxide (v/v 15:15:5:1:0.18).
5. Vortex the glass tubes vigorously for 5 minutes and incubate at 60 °C for 20 min.
6. Pellet the cell debris by centrifugation at 1,800 ×g for 10 min and collect the supernatant in a new glass tube.
7. Dry the supernatant under a stream of nitrogen.
8. Add 1 ml of water-saturated butanol and sonicate for 5 min in a water bath sonicator.
9. Add 500 µl of water and vortex further for 2 min.
10. Centrifuge at 3000 ×g for 5 min.
11. Collect the upper butanol phase, dry it under a stream of nitrogen and store the glass tubes at -20oC till further analysis.
Mass spectrometry analysis
1. Resuspend lipids in 500µl of 50% methanol for lipidomics analysis.
2. 200µl was transferred to glass vials and placed in the Autosampler in the Water nanoacquity UPLC system.
3. Separate lipids on a nanoAcquity UPLC (Waters) equipped with a HSS T3 capillary column, applying a 10 min linear gradient of buffer A (5 mM ammonium acetate in acetonitrile/water 60:40) and B (5 mM ammonium acetate in isopropanol/acetonitrile 90:10) from 10% B to 100% B. Keep conditions at 100% B for the next 7 min, follow it by a 8 min re-equilibration to 10% B. Set injection volume to 1 µL. Set a constant flow rate at 2.5 µl/min.
4. The UPLC was coupled to QExactive mass spectrometer (Thermo) by a nanoESI source (New Objective Digital PicoView® 550).
5. Key MS settings information:
-The source was operated with a spray voltage of 2.9 kV in positive mode and 2.5 kV in negative mode. Sheath gas flow rate was set to 25 and 20 for positive and negative mode, respectively.
-MS data was acquired using either positive or negative polarization, alternating between full MS and all ion fragmentation (AIF) scans.
-Full scan MS spectra were acquired in profile mode from 107-1600 m/z with an automatic gain control target of 1e6, an Orbitrap resolution of 70,000, and a maximum injection time of 200 ms.
- AIF spectra were acquired from 107-1600 m/z with an automatic gain control value of 5e4, a resolution of 17,500, a maximum injection time of 50 ms and fragmented with a normalized collision energy of 20, 30 and 40 (arbitrary units).
-Generated fragment ions were scanned in the linear trap. Positive-ion-mode was employed for monitoring PC and negative-ion-mode was used for monitoring PS and PE.
6. Identity lipid species based on their m/z and elution time. Use a standard mixture comprising PS 10:0/10:0, PE 17:0/17:0, PC 17:0/17:0, PG 17:0/17:0 and PI 12:0/13:0 for deriving an estimate of specific elution times.
Quantification of lipid species
1. Quantify the lipid intensities using the Skyline software2.
2. For each phospholipid, quantify the precursor species (m), cyclopropane species (m+14) and species that appear upon pulse-labeling with deuterated methionine (m+9, m+16, m+23, m+25).
3. Calculate the fraction of labeled headgroups as (m+9 + m+23 + m+25) / (m + m+9 + m+14 + m+16 + m+23 + m+25).
4. Calculate the fraction of labeled cyclopropane species as (m+16 + m+25) / (m + m+9 + m+14 + m+16 + m+23 + m+25).
5. Calculate the fraction of labeled headgroups and cyclopropane, independent of transport , as (m+9 + m+23) / (m + m+9 + m+14 + m+16 + m+23 + m+25) and (m+16) / (m+14 + m+16 + m+23 + m+25), respectively.
6. Calculate the fraction of doubly labeled mass-tagged species (read-out for lipid transport) as (m+25) / (m + m+9 + m+14 + m+16 + m+23 + m+25).