Optimized protocol for the identification of lipid droplet proteomes using proximity labeling proteomics in cultured human cells

• Published in: STAR protocols Vol. 2; no. 2; p. 100579
• Main Authors: Peterson, Clark W.H., Deol, Kirandeep K., To, Milton, Olzmann, James A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.06.2021; Elsevier
• Subjects: Cell Biology; Cell culture; Cell Membrane; Cell separation/fractionation; Cell-based Assays; Proteomics; Protocol; Cell separation/fractionation; Cell culture; Cell Membrane; Cell-based Assays; Proteomics; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100579

Lipid droplets are endoplasmic reticulum-derived neutral lipid storage organelles that play critical roles in cellular lipid and energy homeostasis. Here, we present a protocol for the identification of high-confidence lipid droplet proteomes in a cell culture model. This approach overcomes limitations associated with standard biochemical fractionation techniques, employing an engineered ascorbate peroxidase (APEX2) to biotinylate endogenous lipid droplet proteins in living cells for subsequent purification and identification by proteomics. For complete details on the use and execution of this protocol, please refer to Bersuker et al. (2018). [Display omitted] •Protocol for the identification of high-confidence lipid droplet proteomes•Biotinylation of lipid droplet proteins using APEX2 targeted to lipid droplets•Purification of biotinylated lipid droplet proteins from buoyant fractions•Label-free quantitative proteomics to define lipid droplet proteomes Lipid droplets are endoplasmic reticulum-derived neutral lipid storage organelles that play critical roles in cellular lipid and energy homeostasis. Here, we present a protocol for the identification of high-confidence lipid droplet proteomes in a cell culture model. This approach overcomes limitations associated with standard biochemical fractionation techniques, employing an engineered ascorbate peroxidase (APEX2) to biotinylate endogenous lipid droplet proteins in living cells for subsequent purification and identification by proteomics.