Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Abstract

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analysing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAB/MPN/Mov34 metalloenzyme DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs.

Figure 1. The MALDI-TOF DUB assay.

(a) Workflow of the MALDI-TOF DUB assay. Each of the 42 DUBs was incubated with all eight diubiquitin isomers individually (M1, K6, K11, K27, K29, K33, K48 and K63) for 60 min at 30 °C. The reaction was stopped with 2% TFA and mixed 1:1 with 0.5 μM ¹⁵N-ubiquitin which serves as an internal standard. Subsequently, the analyte is mixed with 2,5 DHAP matrix and spotted onto a 1,536 AnchorChip MALDI target (Bruker Daltonics). Data analysis is performed using FlexAnalysis (Bruker Daltonics). (b) The MALDI-TOF DUB assay shows high sensitivity. Zoomed area (8,520–8,720 m/z) of MALDI-TOF MS spectra for ubiquitin (Ubi) and ¹⁵N-ubiquitin, in the presence of K11-linked diubiquitin are depicted. The limit of detection was determined as 2 fmol of ubiquitin on the target (in the presence of 42 fmol of ¹⁵N-ubiquitin and 146 fmol of K11-linked diubiquitin). Presence of the doubly charged diubiquitin (diubiquitin [M+2H]²⁺) does not compromise identification of the singly charged ubiquitin (see also Supplementary Fig. 2). (c) Linearity and reproducibility of the MALDI-TOF DUB assay. Scatter plot of different concentrations of ubiquitin (10–10,000 nM) shows high linearity over about three orders of magnitude. Interday reproducibility was very high (Supplementary Table 1). Error bars represent s.d. of measurements. a.u., arbitrary unit; intens., intensity.

Figure 2. Characterizing the linkage specificity of DUBs.

Increasing concentrations (0.02–200 ng μl⁻¹) of DUBs were incubated in triplicate with 1.46 μM of diubiquitin of each linkage type (M1, K6, K11, K29, K33, K48, K63 from Boston Biochem, K27 in-house produced) for 60 min at 30 °C and analysed by the MALDI-TOF DUB assay. The amount of monoubiquitin formed by this reaction was determined by MALDI-TOF MS and used to establish the DUB activity for individual diubiquitin isomers which is shown in a gradient of white (0%) to dark red (100%). The data show that DUBs can be grouped into enzymes cleaving specifically one linkage type (group 1), few linkage types (group 2), unspecific (group 3) or inactive enzymes (group 4). For DUB characterization, see Supplementary Figs 3 and 8.

Figure 3. Inhibition profiles of 11 DUB inhibitors and inhibitor candidates.

Eleven different DUB inhibitors and inhibitor candidates were pre-incubated for 35 min at two different concentrations in duplicate (that is, two different experiments) with a panel of 32 DUBs and subsequently the specific substrate was added and incubated for 60 min (30 °C). Inhibition rates are colour coded with strongest inhibition in dark red, the diubiquitin topoisomers used for each DUB are in brackets. BAY 11-7082, NSC 697923 and SJB3-019A show some selectivity at 1 μM against USP7 and USP8, respectively, while PR-619 and HBX 41,108 inhibit strongly a wide range of DUBs even at low concentration. Other proposed inhibitors such as compound 16, L434078, WP1130 and P22077 show low activity and selectivity in this panel.

Figure 4. IC₅₀ analyses of four inhibitors for selected DUBs.

A subset of four inhibitors was chosen to characterize in more detail by determining their IC₅₀ for three DUBs. BAY 11-7082, NSC 697923 and SJB3-019A were chosen as they have some selectivity for one DUB, HBX 41,108 was chosen as it has been proposed as a USP7 inhibitor which is an attractive drug target. Small inhibitor compounds were pre-incubated for 35 min at different concentrations (0–30 or 0–100 μM) in triplicates (that is, three different experiments) and subsequently the specific substrate was added and incubated for 60 min (30 °C). Diubiquitin topoisomers used for each DUB are named on the y axis. Data show that NSC 697923 and BAY 11-7082 inhibit strongly USP7 with IC₅₀<0.2 μM, while HBX 41,108 inhibits it at ~6 μM. SJB3-019A inhibits USP8 and USP2 about 10-fold better than USP1. See also Supplementary Table 4 for P values. Error bars represent s.d. of measurements. For statistical analysis, four parameter logistic curve (best‐fit solution, nonlinear regression-dynamic fitting) and normality tests (Kolmogorov–Smirnov) are used (SigmaPlot, v. 12.5).