Identification of Lipid Droplet Proteomes by Proximity Labeling Proteomics Using APEX2
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.
Key wordsProximity labeling Biotinylation Lipid droplet APEX APEX2 Proteome Organelle
This work was supported by grants from the NIH (R01GM112948 to J.A.O.) and from the American Heart Association (16GRNT30870005 to J.A.O.). J.A. Olzmann is a Chan Zuckerberg Biohub investigator. We thank Clark Peterson for comments on the in-gel protein digestion protocol.
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