Fig. 4: Identification of USP7 substrates in a time-course experiment.

a Schematic of the USP7 inhibitor time-course experiment. HCT116 cells were treated with DMSO or 10 µM of FT671 and harvested in SDC buffer at the indicated time points. After tryptic digestion of 2 mg of total protein per sample, both the ubiquitinome and the proteome were acquired in single-shot DIA mode and the resulting raw files processed using DIA-NN. b Volcano plots of the ubiquitinome (15 min) and the proteome (6 h) after FT671 treatment are shown on the left. Significantly-regulated proteins and K-GG peptides (LIMMA⁵², 5% FDR) are colored (Log2 fold changes −1 < x < 1 in blue and x < −1 or x > 1 in red). The Venn diagram shows significantly and >2-fold ubiquitinated proteins at 15 min (red), and ubiquitinated proteins that were significantly downregulated by more than 20% over the 6 h time course (blue). The overlapping proteins were significantly enriched for categories such as zinc finger and ubiquitin-protein ligase activity. BH FDR = Benjamini–Hochberg false discovery rate. c Heat map of proteins that were significantly downregulated by more than 20% upon FT671 treatment (or more than 50%, in magenta) and that showed significant, and more than twofold induction of at least one ubiquitination site at 15 min. Ubiquitin peptide profiles were averaged and both data were matched based on gene level. Topors was included in the heatmap - its protein intensities were below detection limit (missing values, in gray) because it was rapidly degraded upon FT671 treatment. d Profiles of Log2 fold changes (FT671/DMSO) at different time points for different cell cycle regulators upon USP7 inhibition. Plotted are mean ± SEM. n = 4 biological replicates. e After transfection with different siRNAs (Control, USP7_1, USP7_2) for 48 h, HCT116 cells were treated with DMSO/10 µM FT671 for 5 min. Shown are Log2 fold changes (FT671/DMSO) of all quantified K-GG remnants for siRNAs targeting USP7 (y-axis on both plots) or a control siRNA (x-axes). Selected targets found to be regulated in the time course experiment (Fig. 4a–d) are colored. f Significantly upregulated ubiquitinated peptides at 15 min of FT671 treatment were mapped onto a BioGrid³⁷ network for reported USP7 interacting proteins (filtered for four evidences). The proteins were colored according to the peaking time of the ubiquitin signal (averaged profiles for significantly upregulated peptides). Source data are provided as a Source Data file.