Analysis and Quantification of the Mitochondrial–ER Lipidome

Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood. Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases. Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes. The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions. We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments. This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress. Key features • Analysis and quantification of lipids in mitochondria–ER fraction through liquid chromatography–tandem mass spectrometry-based lipidomics (LC-MS/MS lipidomics). • LC-MS/MS lipidomics provide precise and unbiased information on the lipid composition in in vitro systems. • LC-MS/MS lipidomics facilitates the identification of lipid signatures in mammalian cells.

This protocol is used in: eLife (2023), DOI: 10.7554/eLife.87340Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood.Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases.Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes.The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions.We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments.This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress.

Recipes 1. Cell culture media
DMEM high glucose 1 mM GlutaMax 10% FBS For 500 mL of DMEM high glucose, add 5.5 mL of Glutamax and 50 mL of FBS.

Basal media
DMEM high glucose 1 mM GlutaMax 0.2% BSA with fatty acid For 500 mL of DMEM high glucose, add 5.5 mL of Glutamax and 0.2 g of BSA.

Procedure
We describe below the step-by-step procedure for performing a mitochondria-ER isolation followed by lipidomic analysis and quantification [6].This procedure has been applied to both L6 myotubes and HeLa cells and published in Diaz-Vegas et al. [2].For a different cell line, further optimization might be necessary (e.g., starting material).Store all buffers at 4 °C and perform the procedure on ice.Room temperature is defined as 22 °C throughout this protocol.Washing steps are provided for each step throughout the protocol.For this protocol, 15 fully confluent dishes of 15 cm were pulled together to obtain enough material to isolate the different fractions (equal to one biological replicate).b.Remove the media and wash cells with room-temperature PBS (three times, 6 mL each time).c.Add 10 mL of basal media and incubate at 37 °C for 2 h in the incubator (95% O2, 5% CO2).

Harvesting and cell lysis
a. Transfer the dishes to an ice tray.b.Immediately, wash the cells five times with ice-cold DPBS by gently adding the buffer against the wall of the dish to prevent cell loss.c.After the last wash, add 4 mL of lysis buffer 1 to one plate.d.Using a cell scraper, remove the cells (see Note 2).
Published: Jul 05, 2024 e. Tilt the plate to allow cells to accumulate in one region.When the first plate is ready, remove DPBS from a second plate and transfer the entire volume from plate 1 to plate 2. f.Repeat the scrapping process in the new dish.Continue this procedure until all plates have been scraped, collecting the entire sample (see Note 3).g.Transfer the collected sample to an ice-cold 50 mL conical tube.h.Spin down cells at 1,500 rpm (300× g) for 5 min in a JA-12 rotor at 4 °C.i. Discard supernatant and resuspend cells in 5 mL of lysis buffer 1. j. Assemble the cell homogenizer (see Note 4).k.Equilibrate the cell homogenizer with 1 mL of lysis buffer 1. Discard this buffer after equilibration.l.Transfer cell suspension using a 1 mL syringe and process the samples using the cell homogenizer.m.Pass 10 times back and forth through the cell homogenizer (see Note 5).q.Divide the supernatant into two ice-cold 1.5 mL Eppi ® tubes for isolation of crude mitochondria (reserve 1 mL as whole-cell lysate for further experiments, e.g., lipidomics in whole lysate, western blotting, etc., and store at -80 °C). 4. Crude mitochondria isolation a. Transfer the 1.5 mL Eppi ® tubes to a refrigerated microcentrifuge and spin at 8,500 rpm (10,300× g at rmax) for 10 min in a JA-12 rotor at 4 °C.This will separate crude mitochondria (pellet) from postmitochondrial fraction (supernatant).b.Transfer supernatant containing post-mitochondrial fraction to a new 1.5 mL Eppi ® tubes and store at -80 °C.c.Resuspend pellet in 1 mL of lysis buffer 1 by gently pipetting up and down (see Note 7).

Isolation of mitochondria-ER fractions
a. Carefully, layer the suspension into a polycarbonate tube containing 7.9 mL of 18% Percoll gradient (see Note 8).Add 3 mL of Lysis buffer 1 on top with a p1000 to calibrate the tubes (see Note 9).b.Weigh each polycarbonate tube and balance with Lysis buffer 1 (± 0.001 g) (Note 10).c.Centrifuge at 95,000× g at rmax for 30 min at 4 °C using a sw40Ti rotor.d.With a 1 mL syringe, aspirate the band at the top portion of the tube and transfer it (~2 mL) into two Beckman 1.4 mL tubes (1 mL per tube).This first band will contain the mitochondria-ER fraction.e.With another 1 mL syringe, aspirate the band at the bottom of the tube and transfer it (~2 mL) into four Eppi ® tubes (500 µL per tube).This will contain the pure mitochondrial fraction.f.Weigh each Beckman 1.4 mL tube and balance with Lysis buffer 1 (± 0.001 g).g.Spin down the mitochondrial-associated membranes (MAM) fraction at 60,000 rpm for 1 h using the TLA 110 rotor at 4 °C.h.Mitochondria-ER will be a mucous pellet on a clear Percoll sediment at the bottom of the tube.
Aspirate out Mitochondria-ER carefully with a micropipette and transfer it to an Eppi ® tube (~200 µL) (see Note 11).i. Wash the mitochondria-ER pellet three times with lysis buffer 2 (max speed for 15 min in JA-12 rotor at 4 °C).j.Add 50 µL of 5× sample buffer and adjust the volume to 300 µL with 1× sample buffer for western blotting or store at -80 °C for lipid extraction.k.Add 1 mL of lysis buffer 1 to each tube with pure mitochondria and mix (see Note 7).l.Spin down the mitochondrial fraction at 10,000 rcf for 10 min at 4 °C.m.Mitochondria will form a loose pellet.Carefully aspirate most of the supernatant (~1.2 mL) and replace it with more lysis buffer 1. n. Spin down the mitochondrial fraction at 10,000 rcf for 10 min at 4 °C.o.Aspirate supernatant and resuspend pellet in 300 µL of 1× sample buffer (100 µL per tube) or store at -80 °C for lipid extraction.Resuspension requires repeated up and down pipetting.
e. Quantification of lipid profile in whole lysate and mitochondrial fraction is shown in Figure 1.

Data analysis
Lipids are quantified as the area under the peak for a specific precursor-product ion pair.It is recommended that each peak comprises a minimum of 8-10 scans for that specific precursor-product ion pair.Peak detection and integration is performed with vendor-specific software or freeware such as Skyline [11].For our analyses, TraceFinder was used.The amount of each lipid is determined first as the ratio to its class-specific internal standard.This is then multiplied by the amount of internal standard added to estimate the nmol of each lipid in the sample, then divided by the amount of protein used for lipid extraction, so that lipid levels are expressed as nmol lipid/mg protein.Levels of individual lipid species may then be compared between sample groups or treatment conditions using statistical analyses appropriate to the experimental design.

Validation of protocol
This protocol has been used and validated in the following research article(s): • Diaz-Vegas et al. [2], Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle, eLife 12, RP87340 (Figure 2, panel C, D; Figure 3, panel C, D).Ceramide species were quantified using targeted lipidomics.N = 3 biological replicates.Control conditions refer to cells with vehicle control for each experiment.Student's t-test was utilized for comparing two groups, whilst ordinary one-way ANOVA followed by Dunnett's multiple-comparison test was employed for comparing multiple groups.
n. Transfer lysate to a new ice-cold 50 mL Falcon tube.Repeat step A3m until all samples are collected in this new Falcon tube (see Note 6).o.Spin down nuclei for 10 min at 1,810 rpm (600× g at rmax) in a JA-12 rotor at 4 °C.p. Transfer supernatant (~4 mL) to a new ice-cold 50 mL Falcon tube and discard pellet.