Global analysis of the Hsp90-dependent proteome.

(A) Overall experimental design, that shows the steps involved in determining relative quantities of proteins between N and Rc cells, un- or treated with Hsp90 inhibitors, allowing to compare eight samples simultaneously. In LC-MS/MS plot the 8 color peaks express the 8 report ions of iTRAQ. The height peaks show the relative amount of peptides per sample, other black peaks are the MS/MS ion peaks of peptide fragmentations for protein identification. (B) Venn diagrams highlighting the number of proteins depleted in N versus Rc cells treated with three different inhibitors and the GO analysis of those sets of proteins; for GA, the % of proteins in the overlap is indicated, and for all three inhibitors, they are shown in panel C. (C) Protein intersections between N and Rc cells treated with the mentioned Hsp90 inhibitors; the data are from panel B and S3 Fig. (D) Principal component analysis (PCA) of the quantitative proteomic changes of the 656 proteins common to 3 datasets of Rc cells treated with Hsp90 inhibitors. The PCA “loadings” (colored arrows) illustrate the similar/distant behaviors between variables (inhibitors). (E) Proteomic signatures of Hsp90 inhibitors. Heat map depicting the proteins that are up- or downregulated and clustered by common enrichment and depletion signatures for the 3 datasets (top and bottom panels) or distinctive depletion signature for each inhibitor (intermediate panels) on Rc cells. (F) GO functional enrichment analysis of common (or inhibitor-exclusive) affected protein abundance (depletion or enrichment) of Rc treated cells.