Abstract
Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues.
Highlights
One of the most pressing issues in eukaryotic cell biology is the need to determine the dynamic composition of organelles that allow for the coordinated execution of many different
We describe a quantitative proteomics approach based on lipid droplets (LDs) purification and mass spectrometry (MS) that employs SILAC and protein correlation profiles (PCPs), similar to what has been employed for other organelles [29], to efficiently determine an LD proteome with a high degree of confidence
The combination of a careful purification scheme and PCP methodology enabled us to extract from the complete LD-containing fraction a set of just over 100 LD proteins with significant confidence
Summary
Drosophila S2 Cells—Drosophila S2 cells were cultured in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics (100 unit/ml penicillin and 100 g/ml streptomycin) at 27 °C as described elsewhere [34]. The supernatant was adjusted to 1 M sucrose in buffer, and the resulting 2 ml fraction was layered under a 12-ml sucrose step-gradient with 2-ml steps (0.75, 0.5, 0.25, 0.125, 0 M) and centrifuged for 12 h at 200,000g at 4 °C in 13.2-ml thin-wall polyallomer tubes (Beckman Coulter) with a TH-641 swinging bucket rotor in a Sorvall WX80 ultracentrifuge (Thermo Scientific). The resulting peptide mixtures were diluted with 200 l 50 mM ammonium bicarbonate and digested with 4 g sequencing-grade modified trypsin (Promega) per protein overnight at room temperature. Eluted peptides were analyzed using a nanoflow high-pressure liquid chromatography system (Agilent Technologies or Proxeon Biosystems) coupled on-line via a nanoelectrospray ion source (Proxeon Biosystems) to an LTQ-Orbitrap mass spectrometer (Thermo Scientific). Soft clustering was performed using Mfuzz, a software package implemented in the statistical program language R [41]
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