Identification of Lipid Droplet Proteomes by Proximity Labeling Proteomics Using APEX2

Kirill Bersuker; James A Olzmann

doi:10.1007/978-1-4939-9537-0_5

Identification of Lipid Droplet Proteomes by Proximity Labeling Proteomics Using APEX2

Methods Mol Biol. 2019:2008:57-72. doi: 10.1007/978-1-4939-9537-0_5.

Authors

Kirill Bersuker¹, James A Olzmann^{2

3}

Affiliations

¹ Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA.
² Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA. olzmann@berkeley.edu.
³ Chan Zuckerberg Biohub, San Francisco, CA, USA. olzmann@berkeley.edu.

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.

Keywords: APEX; APEX2; Biotinylation; Lipid droplet; Organelle; Proteome; Proximity labeling.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Ascorbate Peroxidases / chemistry
Biotinylation
HEK293 Cells
HeLa Cells
Humans
Lipid Droplets / chemistry*
Lipid Droplets / metabolism
Mice
Proteomics / methods*
Staining and Labeling / methods*

Substances

Ascorbate Peroxidases

Grants and funding

R01 GM112948/GM/NIGMS NIH HHS/United States