Figure 2

Identification of APOA4 interactome in mouse gonadal fat tissues using mass spectrometry. Wildtype 129X1/SvJ male and female mice fasted for 5 h and i.p. injected with a single dose of r-m-APOA4 (2.5 μg/g body weight). Two hours post injection, mice were euthanized and total protein was isolated from the epididymal and periovarian fat tissues (n = 5–6/sex). IP was performed from pooled samples (10 mg total protein per IP experiment) using anti(α)-APOA4-specific and goat IgG (negative control) antibodies in parallel pull-down experiments. Protein eluates were electrophoresed on SDS-PAGE and visualized by (A) Silver and Coomassie blue–stained gel of IPs showed several bands enriched in α-APOA4 pull-downs, but not in goat IgG. Silver-stained lanes from the same gel are shown. α-APOA4 and IgG lanes were excised and processed for mass spectrometry. (n = 5–6/condition for IP and 4/condition for MS). (B) Volcano plot showing relative abundances of the proteins found in APOA4 Co-IP experiments. X axis shows the log 2 of the relative abundances over IgG control pull downs. Y axis the significance as – log₁₀ of P values. The proteins that lie within the red thick dotted line in the volcano plot were plotted in network generated in (D). The right panel shows data as scatter plot of relative enrichment of APOA4-associated proteins vs. IgG pull-downs. Intervals of confidence for 95% (blue lines) and 99.7% (red lines) are indicated. HSDL2 hydroxysteroid dehydrogenase like 2; ALDH1a1/a7 aldehyde dehydrogenase 1 family members A1and A7. (C) Spectral analysis: High-energy collision dissociation–tandem mass spectra obtained from precursor ions with mass 483.2320⁺³ (APOA4) and 687.8901⁺² (LRP1) found in tryptic digests of immunoaffinity pull-downs of anti-APOA4 corresponded to peptides spanning residues L66 to K79 and Y2001 to R2012 of APOA4 and LRP1, respectively. b- and y-type ion series are labeled. (D) Portion of the STRING interactome in the neighborhood around APOA4 highlighted within the red box in (B). The nodes are colored by the Log2 ratio of APOA4 Co-IP/IgG control, except for APOA4, which is colored grey, and the edge widths represent the STRING confidence score. The colored slices around the outside of the nodes represent the enrichment as provided by the Cytoscape stringApp, filtering for only KEGG Pathways (KEGG), GO Cellular Component (GCC), and GO Biologic Process (GBP). The nodes HSDL2, LRP1, and APOA4 are enlarged to correspond with highlighted nodes in the scatter plot shown in 2B. The label color for HSDL2 was changed to white to enhance visibility.