384 pin tool

All chemicals and reagents were purchased from Sigma Aldrich and used without further purification. Stock solutions (0.1 M) were prepared in appropriate solvents as indicated. Master 384-well microtiter plates were prepared either by hand pipetting appropriate mixtures of chemicals or by robotic pipetting with a Biomek FX liquid handling system. Once the master plate was prepared, a 384-pin tool from V&P Scientific, Inc. was used to deliver mixtures from the master plate to the DESI-MS substrate. Transfer was repeated multiple times in order to generate the desired titer plate density (1536-well, 6144-well, etc.). Details for each individual experiment are provided in the ESI.†

The pre-reaction mixture one consisted of 40 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2 mM DTT, 5 mM ATP, 20 nM E1, 350 nM E2 (UbcH5), 25 nM HA-tagged Mdm2, 200 nM MdmX. First, 10 μl of the pre-reaction one was dispensed in each well of 384-well plate (Multiflo, Biotek, Winooski, VT, USA). Then compounds from a chemical library (DIVERSet, ChemBridge) were added in a volume of 8 nl of each by the robot pin tool (PerkinElmer JANUS, Waltham, MA, USA, V&P Scientific 384 Pin tool, San Diego, CA, USA). The reaction was started by adding premixture two, which consisted of 250 nM HA-tagged ubiquitin and 50 nM ubiquitin cryptate at 2 μl per well. After incubation at 37 °C for 1.5 h, the reaction was terminated by adding 10 μl of the detection buffer, which contains 50 mM phosphate buffer pH 7.0, 0.1% BSA, 0.1 M EDTA, 0.8 M KF and 20 nM XL665-conjugated antibody against HA tag. The reaction was kept for 1 h at room temperature before measuring the FRET signal. For the FRET measurement in Perkin Elmer Envision 2103 Multilabel Reader, there is a 100 μs time delay between the excitation (320 nm) and measurement at two different wavelengths (615  and 665 nm), then calculating the ratio for each well individually (ratio=665 nm/615 nm x10⁴). The 10⁴ multiplying factor is introduced for convenient data processing.

The primary screening was carried out in a fully automated robotic platform Cell::Explorer (Perkin Elmer) in a 384-well format. On the day of the experiment, reporter cells were harvested and resuspended into phenol red-free DMEM culture media to obtain a concentration of 200 cells/μl. Twenty-five microliters aliquots of the cell suspension were dispensed to white polystyrene 384-well plates (Corning) by a Multidrop Combi liquid dispenser (Thermo Scientific). The test compounds were transferred from the 384-well compound plates to 384-well assay plates in Janus Automated Workstation (Perkin Elmer) integrated to Cell::Explorer and equipped with 384 Pin tool (V&P Scientific). The final concentration of the screened compounds was 1 μM. Each plate was shaken for 30 s using a plate shaker Variomag (Thermo Scientific) and then incubated at 37°C and 5% CO₂ in humidified atmosphere for 48 h.