Fig. 4: Mass spectrometry-based proteomics identifies TAK-285 cellular protein targets.

a Schematic illustration of experimental design for global proteomics profiling. MCF-7 neoHER2 cells were treated with DMSO, TAK-285 (100 nM or 1 µM), 17-AAG (1 µM), or pretreated with TAK-285 (100 nM or 1 µM), followed by 17-AAG (1 µM). 17-AAG treatment time is 8 h (for the 100 nM set) and 6 h (for the 1 µM set). b Volcano plot depicting the fold-change in protein abundance in MCF-7 neoHER2 cells treated with 17-AAG (8 h) relative to DMSO samples. Each circle represents an individual protein with the color representing protein class (kinase: red; others: blue). X-axis: log2 transformed ratio of the protein levels in the 17-AAG treated samples and the DMSO treated samples. Y-axis: −log10 transformed p-value (dotted lines indicate p = 0.05 and p = 0.01; two-tailed student’s t-test). Density curves for the two protein groups are shown at the bottom. c Distribution of ratio changes between 17-AAG (8 h) and DMSO in protein groups: by protein function, HSP90-interaction, or HSP90 dependency (p < 0.00001 (***)) by Welch two-sample t-test (box plots show minimum, first quartile, median, third quartile, and maximum). d, e Volcano plot showing the fold change in protein abundance in MCF-7 neoHER2 cells treated with a combination of 17-AAG and TAK-285, 100 nM d or 1 µM e relative to 17-AAG only samples. Annotated proteins (and alternative gene names) include: ERBB2 (HER2), PHLA1 (PHLDA1), PHLA2 (PHLDA2), KPCD2 (PRKD2) and EGFR. f Quantitation of ERBB2 (HER2) protein relative abundance in the four treatment groups. g, h Relative abundance of PHLDA1 g and PHLDA2 h proteins in the four treatment groups (1 µM TAK-285). Individual data points in F-H are calculated as percentage of sum total TMT signal for the protein (across all treatment groups), and shown as mean with standard deviation indicated. The statistical significance of the relative abundance of each protein in the different samples were determined using two-tailed student’s t-tests for TAK-285 vs. DMSO, 17-AAG vs. DMSO, and TAK-285 + 17-AAG vs. 17-AAG (n = 2 for TAK-285 alone; n = 3 for all other conditions; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Source data are provided as a Source Data file.