Abstract
Lipid droplets (LDs) are ubiquitous lipid storage organelles composed of a neutral lipid core surrounded by a phospholipid monolayer that is decorated with integral and peripheral proteins. Accurate identification of LD proteins using biochemical fractionation methods has been challenging due to the presence of contaminant proteins from co-fractionating organelles. Here, we describe a method to identify high-confidence LD proteomes that employs an engineered ascorbate peroxidase (APEX2) to induce spatially and temporally restricted biotinylation of LD proteins. This proximity labeling method can be broadly applied to define the composition of the LD proteome in any cultured cell line and can be utilized to examine LD proteome dynamics.