Figure 6.
Protein correlation profiling identifies LD-enriched proteins from brain tissue of DDHD2−/− mice. (A) Schematic representation of the protein correlation profiling experiment. DDHD2−/− mouse brains were homogenized and sequentially fractionated by differential centrifugation and a sucrose density gradient centrifugation. Proteins from all fractions were precipitated and processed in parallel for mass spectrometric analysis. Spectral counts were normalized for each quantified protein across fractions to generate the correlation profiles. (B) Reproducibility of spectral count values for proteins in each fraction from biological replicate experiments. The numbers of spectral counts observed for each protein in experiments 1 and 2 are plotted on the X- and Y-axes, respectively. Pearson correlation coefficients between all pairwise groups of fractions are reported in Figure S7C. Protein correlation profiles for representative organellar marker proteins: PLIN2, lipid droplets (LDs); CALR, endoplasmic reticulum (ER); GAPDH, cytosol; VDAC2, mitochondria; ATP1A1, plasma membrane; HIST1H2B, nucleus. Data represent average values ± SEM (N = 4 biological replicates). (D) Representative LD-enriched proteins in brain tissue from DDHD2−/− mice, defined as proteins with ≥3-fold spectral counts in fraction 1 vs the fraction with the next highest spectral counts that also met the following filtering criteria: ≥15 total spectral counts and detection in fraction 1 in at least three of the four biological replicates. Data represent average values ± SEM (N = 4 biological replicates). (E) Hierarchical clustering of proteins based on relatedness of correlation profiles across fractions. Average spectral count values for each protein in each fraction were normalized to the fraction with the most spectral counts for that protein. The horizontal brace highlights the lipid droplet (LD)-enriched cluster that contains 49 members.