Figure S2.
Assessment of Reproducibility of Biological Replicates and Technical Validation of the Quantification, Related to Figure 2
(A–E) Samples were analyzed on a Q-executive (using MS2 for reporter ion quantification) as well as on a Fusion Lumos (using SPS MS3 for reporter ion quantification) mass spectrometer to assess the reproducibility of biological replicates and as a technical validation of the quantification.
(A) Annotated MS2 and MS3 spectra for the BRD2 peptide VVHIIQAR.
(B) The first two scatterplots compare protein fold changes (FC) between two biological replicates using MS2 and MS3 based quantification respectively, the third scatterplot compares fold changes calculated using either MS2 or MS3 based quantification performed on aliquots from the same samples. The density plots show the difference of protein fold changes between the biological replicates in the first and second plot and the difference between protein fold changes from MS2 and MS3 based quantification in the third plot.
(C) Protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 24 h with 10 μM JQ1-VHL PROTAC relative to vehicle, for BRD2, BRD3 and BRD4 using MS2 and MS3 quantification in two biological replicates.
(D) Protein fold changes (FC) observed in mature (upper panel) and nascent (lower panel) forms of proteins in THP-1 cells treated for 24 h with 10 μM JQ1-Az relative to vehicle, for BRD2, BRD3 and BRD4 using MS2 and MS3 quantification in two biological replicates.
(E) Scatterplots showing protein fold changes (FC) observed in mature forms (upper panel), nascent forms (middle panel) and the total proteome level estimated from the summed-up reporter ion abundances over mature and nascent proteins (lower panel) of proteins in THP-1 cells treated for 24 h with JQ1-Az (10 μM), JQ1-VHL PROTAC (1 and 10 μM) or VHL alkyne (10 μM) relative to vehicle treated cells. Red closed circles indicate statistically significant regulation (p < 0.05) and the dashed diagonal indicates the equality line. N indicates the number of proteins robustly quantified in both replicates.