Quantification of Phosphatidylinositol Phosphate Species in Purified Membranes

Phosphoinositide lipids (PIPs) are required for various processes during macroautophagy, such as phagophore formation and autophagosome-lysosome fusion. Hence, quantification of the seven PIP species in autophagosome membranes is an important tool to understand how these lipids govern the transition of autophagosomes into autolysosomes. Here, we describe microscopic and mass spectrometry methods which, although designed to quantify the different PIP species on purified lysosomes, can also be applied to analyze autophagosomal PIPs.