Impact 2 q tof spectrometer

Ubiquitination reaction samples (3 µg protein) were diluted in denaturing buffer (3 M urea, 25 mM TEAB pH 8.5, 0.5 mM EDTA) and reduced using 2 mM TCEP for 10 min at 37°C, followed by alkylation with 50 mM chloroacetamide for 30 min at room temperature in the dark. Chloroacetamide was used to avoid iodoacetamide-induced artefacts that mimic ubiquitination [84 (link)]. Samples were diluted with 50 mM TEAB pH 8.5 to 1 M urea and digested with 0.5 µg trypsin (Sigma) for 3 h at 37°C. Digested peptides were purified using C18 Spin Columns (ThermoFisher) and resuspended in 0.1% formic acid. Peptides (0.5 µg) were captured and eluted from an Acclaim PepMap100 C18 column with a 2 h gradient of acetonitrile in 0.1% formic acid at 200 ml min⁻¹. The eluted peptides were analysed with an Impact II Q-TOF spectrometer equipped with a Captive Spray nanoelectrospray source (Bruker). Data were acquired using data-dependent automatic tandem mass spectrometry (auto-MS/MS) and analysed with MaxQuant using a standard search procedure against a custom-made FASTA file including GST-Ub, Parkin, Ub and UbcH7. Methionine oxidation, Lys ubiquitination (diGly) and Ser/Thr phosphorylation were included as variable modifications. Cysteine carbamylation was included as fixed modification.

One milligram of the candidate compound was dissolved in 2 mL of methanol. About 3 µL of this solution was injected with a Dionex UltiMate 3000 autosampler into a 300 μm × 15 cm HPLC C18 column (2 μm, 100 Å Acclaim PepMap; Thermo Fisher Scientific). The HPLC system was the Dionex UltiMate 3000 RSLCnano system. The mobile phase was water with 0.1% formic acid (A) and methanol (B). The flow rate of the loading pump was 25 µL/m, and of the NC pump was 5 µL/m. The gradient was 5% B initially, reaching 99% B at 35–45 min, 99% B at 45–50 min, and 5% B at 55–60 min. The mass spectrometry data were analyzed with a Bruker Daltonics Impact II QTOF spectrometer (in positive mode). The gas temperature was 200 °C. The drying gas was nitrogen with a flow rate of 4 L/min. The nebulizer was at 0.3 bar.