Recent studies have illuminated novel roles of lysosomes that go far beyond simple catabolism and function in the coordination of cellular metabolism and signaling. Promising therapeutic strategies emerge from knowledge in the molecular mechanisms and physiological roles of lipid metabolism in lysosomes. Global monitoring of the function and dysregulation of lysosomal lipid metabolism requires a methodology that resolves the complexity of lysosomal lipidome by quantitatively detecting hundreds of lipid species of diverse physicochemical properties. We describe here a detailed protocol that couples isolation of superparamagnetic iron dextran-loaded lysosomes from cultured mammalian cell lines with quantitative mass spectrometry-based shotgun lipidomics.
Keywords: Lipids; Lysosomes; Mass spectrometry; Metabolism; Quantitative profiling; Shotgun lipidomics.