Lipid droplet-targeted APEX proteome
Lipid droplet (LD) function is regulated by a complement of integral and peripheral proteins that associate with the LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to U2OS and Huh7 cells identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins.
Sample Processing Protocol
U2OS and Huh7 cells expressing a V5-tagged APEX2 fused to either PLIN2 or an inactive mutant of ATGL were cultured in DMEM containing 4.5 g/L glucose and L-glutamine (Corning) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific and Gemini Bio Products) at 37°C with 5% CO2. SILAC isotope labeling was performed by growing cells for at least 6 days in DMEM lacking arginine and lysine (Corning) supplemented with 10% dialyzed FBS and the appropriate SILAC amino acids (Cambridge Isotope Laboratories, Inc.): light (K0R0), L-Lysine (Lys0) and L-Arginine (Arg0); medium (K4R6), 4,4,5,5-D4-L-lysine (Lys4) and 13C6-L-arginine (Arg6); heavy (K8R10), 13C615N2-L-Lysine (Lys8) and 13C615N4-L-arginine (Arg10). For each APEX2 cell line, 18 15-cm plates of cells were treated with 5-10 ng/µl doxycycline for 48 h followed by incubation in 200 μM oleate and 7 μM Hemin for 24 hr. Cells were subsequently treated with 500 μM biotin-phenol for 30 min. Prior to harvesting, biotinylation of proteins was catalyzed by addition of 1 μM H2O2 for 1 min, and the reaction was quenched by washing cells 2X with PBS containing 10 mM sodium ascorbate and 5 mM Trolox. Cells were harvested in PBS, centrifuged for 10 min at 500 x g, and cell pellets were incubated for 10 min in cold hypotonic lysis medium (HLM) containing cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich). Cells were dounced 80X strokes in a 7 ml dounce and lysates were centrifuged at 1000 x g for 10 min. The supernatant was subsequently transferred to Ultra-Clear ultracentrifuge tubes (Beckman-Coulter), diluted to a final concentration of 20% sucrose/HLM, and overlaid by 4 ml of 5% sucrose/HLM followed by 4 ml of HLM. Overlaid samples were centrifuged for 30 min at 15,000 x g in an ultracentrifuge using an SW41 swinging bucket rotor (Beckman-Coulter). Buoyant fractions were isolated using a tube cutter (Beckman-Coulter), additional fractions were pipetted from the top of the sucrose gradient in 1 ml increments and pellets were resuspended in 1 ml HLM. All fractions were brought to a final volume of 1% SDS and sonicated. Buoyant fractions were additionally incubated at 37°C for 1 hr with sonication every 20 min, followed by a final incubation for 10 min at 65°C. For isolation of biotinylated proteins from U2OS cells, buoyant fractions containing 1% SDS were diluted with PBS/0.1% Tween-20 (PBST) to a final concentration of 0.1% SDS. 0.4 ml of streptavidin-conjugated agarose bead slurry (Thermo Fisher Scientific) was washed 3X with PBST and added to the diluted buoyant fractions for 4 hr at RT with constant mixing. Beads were centrifuged at 2000 x g and washed 5X with PBST, followed by 3X washes with PBS and 3X washes with 50 mM ammonium bicarbonate. The beads were resuspended in one bead volume of 50 mM ammonium bicarbonate containing 0.02% Rapigest (Waters), heated at 65°C for 15 min and bound proteins were digested O/N at 37°C with 1 μg mass spectrometry grade trypsin (Promega). After protein digestion, beads were removed and the supernatant was acidified to pH < 2 by addition of 500 mM HCl and incubation at RT for 45 min. All precipitated material was removed by centrifugation at 20,000 x g for 15 min. Peptides were dried down to a final volume of 15-20 μl in a vacuum centrifuge. For isolation of biotinylated proteins from Huh7 cells, an in-gel digestion protocol was used to minimize contamination of samples with streptavidin from the beads. Buoyant fractions containing 1% SDS were diluted with HLM buffer to a final concentration of 0.1% SDS. 0.2 mL of streptavidin-conjugated agarose bead slurry (Thermo Fisher Scientific) was washed 3X with PBST and 1X with HLM buffer and added to the diluted buoyant fractions for 4 hr at RT with constant mixing. Beads were centrifuged at 2000 x g and washed 5X with PBST, followed by 3X washes with PBS. Proteins were eluted with 2% SDS + 3 mM biotin by incubating at RT for 15 min with constant mixing followed by heating at 95°C for 15 min. The eluted proteins were mixed with 1X sample dye and run into a mini-PROTEAN TGX 4-20% polyacrylamide gel (Bio-Rad), and proteins were digested in-gel overnight with 0.5 μg trypsin in 5% acetonitrile/5 mM ammonium bicarbonate. Digested peptides were extracted by addition of 5% formic acid in acetonitrile and incubation at 37°C for 15 min with constant agitation. The resulting supernatant was dried down to a final volume of 15-20 μL in a vacuum centrifuge. Total proteins from U2OS and Huh7 buoyant fractions were isolated by dilution of fractions to a final volume of 1% SDS and addition of trichloroacetic acid (TCA) to a final concentration of 15%. Precipitated proteins were pelleted by centrifuging at 20,000 x g for 30 min at 4°C, washed twice with cold acetone and resuspended in 0.02% Rapigest.
Data Processing Protocol
1 μg of peptides was analyzed by LC-MS/MS on a Thermo Scientific Q Exactive Orbitrap Mass spectrometer connected to a Proxeon Easy-nLC II HPLC (Thermo Fisher Scientific) and Proxeon nanospray source at the University of California, Davis Proteomics Core Facility. Peptide identity and MS/MS counts were determined by analyzing RAW output files in MaxQuant (Max Planck Institute of Biochemistry) using the reviewed human protein database obtained from UniProt. Variable modifications were set to include N-terminal acetylation and oxidation. The FDR was set to 1% and minimum peptide length was set to 6 amino acids.
Bersuker K, Peterson CWH, To M, Sahl SJ, Savikhin V, Grossman EA, Nomura DK, Olzmann JA. A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes. Dev Cell. 2018 44(1):97-112.e7 PubMed: 29275994