Fig 1: Optimizing the ABPP-HT workflow. (A). Number of DUBS identified by timsTOF MS with increasing amounts of HA-Ub-PA-labelled MCF-7 lysate protein, after immunoprecipitation and elution with 0.15% TFA. (B). Western blot densitometry quantification (full blot in Figure S2D) of USP7 in the immunoprecipitation loading flow-through, with increasing amounts of HA-Ub-PA-labelled MCF-7 lysate protein quantity immunoprecipitated and eluted with 0.15% TFA. (C). Number of DUBs identified by LC-MS/MS with different IP elutions: 0.15% TFA, 6 M urea, HEPES, * = On column digestion. (D). Log2 intensities of DUBS identified with different elution methods by a QE orbitrap MS. 0.15% TFA, 6 M urea, HEPES, * = On column digestion. (E). Log2 intensities of DUBs identified with different elution methods by a timsTOF MS. 0.15% TFA, 6 M urea, 5% SDS. F. USP30 immunoblots showing mouse brain lysate displacement of a covalent (3-b) and non-covalent (39) USP30 inhibitor with increasing HA-Ub-PA (at 37°C) incubation times. (G). The densitometric quantification of Figure 3F from the intensity of the HA-Ub-PA-labelled band, normalized to the intensity of both USP30 bands together. (H). timsTOF DUB intensities of MCF-7 labeled with HA-Ub-PA for 10 min normalized to 45 min at 37°C (SEM, n = 3). I. Optimization workflow for high-throughput DUB inhibitor screening using ABPP LC-MS/MS.
Fig 2: ABPP-HT reveals DUB inhibitor selectivity and specificity compatible with higher throughput. (A). The activity of a panel of DUBs from MCF-7 identified from timsTOF MS, in response to USP7 specific inhibitors FT671 (n 3 (for 0.2 µM n 2)), FT827, HBX108 and P22077. (B). The activity of a panel of DUBs from mouse brain lysate identified from timsTOF MS, in response to USP30 specific inhibitors 3-b and 39. (C). The activity of a panel of DUBs in MCF-7 lysates identified by timsTOF LC-MS/MS, in response to the cysteine modifier NEM, and broad spectrum DUB inhibitor PR619 (PR619 n 2). D-I. From left to right concentration dependences of USP7 from inhibitors FT671, FT827, HBX41108, and P22077 in MCF-7 lysates, and USP30 inhibitors for 3-b and 39 in mouse brain. (J). IC50 values extracted from D-I, fit to equation: Y 100/(1 + X/IC50). * normalized raw intensities, not LFQ intensities.
Fig 3: USP30 promotes c-Myc deubiquitination. A Interaction between USP30 and c-Myc in HSC4 cells. c-Myc expression in B HSC4 and C SCC4 cells with or without USP30 knockdown and overexpression. c-Myc levels in HSC4 cells with USP30 knockdown with or without D protease inhibitor MG132 (10 μM) or E CHX treatment. F Effect of USP30 overexpression on c-Myc ubiquitination in SCC4 cells transfected with WT-USP30, C77S-mutant USP30, or vector. G Effect of USP30 knockdown on c-Myc ubiquitination in HSC4 cells
Fig 4: USP30 overexpression increased SCC4 cell viability and glutamine consumption, and inhibits apoptosis. SCC4 cells were transfected with WT-USP30, C77S-mutant USP30, or vector, and (A) USP30 expression, (B) cell viability, (C, D) apoptosis, (E) glutamine consumption, and (F) expression of c-Myc, GLS1, and SLC1A5 were measured. *P < 0.05, ***P < 0.001
Fig 5: USP30 upregulation is associated with poor prognosis. A mRNA and B protein levels of USP30 in stage 1 or 2 OSCC tissues (I/II), stage 3 OSCC tissues (III), and adjacent nontumor tissues (N). C USP30 protein levels in human OSCC tissue microarrays detected by IHC (scale bars: 100 µm). D Overall survival rate analysis. E USP30 expression in human OSCC cell lines and oral epithelial cell HOEC. **P < 0.01, ***P < 0.001
Supplier Page from Abcam for Anti-USP30 antibody