Protein correlation profiles identify lipid droplet proteins with high confidence

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.

Fig. 1.

Experimental scheme of quantitative analysis of an LD proteome via protein correlation profiling. LDs are purified from two populations of Drosophila S2 cells, one unlabeled (Arg0Lys0) (purple) and one labeled with heavy amino acids Arg10 and Lys8 (green). After three steps of differential centrifugation (centrifugation pellets are fractions 9–7), cell lysates are further fractionated with a sucrose step gradient (fractions 6–1). LDs are large round structures in fraction 1. The partitioning of schematic sample proteins is indicated. For quantitative analysis, the top (LD-containing) fraction of the light sample is mixed with each of the six fractions of the sucrose gradient and the three pellet fractions of the heavy samples. Mixed fractions are de-lipidated, and proteins are precipitated. After in-solution digestion of the proteins, samples are separately analyzed via liquid chromatography on-line coupled to high-resolution MS/MS. The heavy/light ratio (H/L) for the proteins of each fraction was normalized by dividing it by the highest ratio among the fractions, giving a value of 0 to 1 (H/L) for each fraction. Profiles show values plotted against each fraction for a number of example proteins represented schematically.

Fig. 2.

High reproducibility of MS measurements for the LD PCP. The ratios of the heavy and light labeled versions of each protein from two independent measurements are plotted against each other for each fraction of the purification. The high correlation of the data points in the scatterplots indicates that differences in the amounts of proteins from light versus heavy labeled samples can be detected with high reproducibility. Pearson correlations and regression lines are shown in red.

Fig. 3.

Identification of LD proteins in S2 cells via LD PCP. A, averaged fractionation profiles for different organelle marker proteins. The average normalized ratios for marker proteins of the indicated cellular organelles, as determined via quantitative LC-MS/MS, are shown for fractions of an LD purification. Values are mean ± S.D. of five proteins. B, hierarchical clustering of H/L for proteins identified in fraction 1 of LC-MS/MS analysis of all fractions of a LD purification with Perseus (39). At least five of nine valid values per protein were required. The color code represents the protein ratio H/L normalized by the maximum value among the fractions. Gray indicates that the protein was not identified in that fraction. Clusters enriched for proteins of certain organelles are marked with white boxes. Cellular organelles whose proteins were enriched in a certain cluster are indicated on the right. C, soft clustering of proteins identified in fraction 1 of LC-MS/MS analysis of all fractions of an LD purification with Mfuzz in R (41). Proteins identified in the LD fraction that were identified in at least five of the nine fractions were analyzed. A normalized H/L was used. The cluster number C was set at 9, and cluster stability m = 1.75. Clusters that showed enrichment of proteins of a certain organelle or function are indicated. Proteins with a minimal membership value of 0.3 for LD cluster 1 are shown in supplemental Table S2.

Fig. 4.

Localization of a subset of proteins found in the LD proteome as determined via fluorescent microscopy of expressed proteins. The indicated fluorescent mCherry-tagged enzymes were transiently expressed in S2 cells (left-hand panels, red) loaded with 1 mm oleate for 12 h. LDs were stained with BODIPY (middle panels, green). The overlays of the two channels and zoomed views of a representative LD section are shown (rightmost two panels). Bar = 5 μm (overview) or 1 μm. Protein correlation profiles for the tagged proteins are shown in the right-hand panels in red. Dark gray lines indicate averaged PCPs for LD proteins, and bright gray lines indicate averaged protein correlation profiles for ER proteins (Fig. 3A). A, proteins localizing exclusively to LDs. B, proteins localizing to LDs and ER. C, proteins localizing to LDs and any other cellular compartment.

Fig. 5.

Comparison of the LD PCP with other proteomic studies and analysis for enriched pathways. A, comparison of the LD proteome with published Drosophila LD proteomes (10, 11). Venn diagram shows overlap between the three studies. B, overrepresentation of specific pathways among LD-protein-associated functions. Groups of proteins with related functions detected on LDs are shown.

Fig. 6.

Several N-glycan biosynthesis enzymes localize to LDs. A, the indicated mCherry-tagged proteins of the N-glycan synthesis pathway were expressed and analyzed as described for Fig. 4. PCPs of the indicated proteins are shown on the left. B, organization of the N-glycan biosynthesis on LDs and ER.