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. 2024 Jul 5;14(13):e5028.
doi: 10.21769/BioProtoc.5028.

Analysis and Quantification of the Mitochondrial-ER Lipidome

Alexis R Diaz-Vegas  1 , Anthony S Don  2 , James G Burchfield  1
Affiliations

Analysis and Quantification of the Mitochondrial-ER Lipidome

Alexis R Diaz-Vegas et al. Bio Protoc. .

Abstract

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.

Keywords: Cardiolipin; Ceramides; Endoplasmic reticulum; Lipidomics; Mitochondria; Subcellular fractionation.

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Conflict of interest statement

Competing interestsThe authors have no competing interests to declare.

Figures

Figure 1.
Figure 1.. Representative results from targeted lipidomic for ceramides.
A. L6 myotubes were incubated with the fatty acid palmitate (150 µM for 16 h) and ceramide levels were measured in whole lysate or mitochondrial–ER fraction. B. Abundance of the different ceramide species in lysate and mitochondrial–ER fraction. N = 3–4 biological replicates, **p < 0.001, ****p < 0.0001.
See this image and copyright information in PMC

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