Q tof 6530 mass spectrometer

Fluorescence intensity measurements were used to monitor the cleavage of a Ub–rhodamine (Ub-Rho110) substrate. All activity assays were performed in black 384-well plates in 20 mM Tris–HCl, pH 8.0, 150 mM potassium glutamate, 0.1 mM Tris(2-carboxyethyl)phosphine) (TCEP), and 0.03% bovine gamma globulin with a final assay volume of 20 μl. Compound IC₅₀ values for DUB inhibition were determined as previously described (18 ). Briefly, an 11-point dilution series of compounds were dispensed into black 384-well plates using an Echo-550 Acoustic Liquid Handler (Beckman Coulter). USP30, 0.2 nM (residues 64–502Δ179–216 & 288–305; Viva Biotech [Shanghai] Ltd), was added, and the plates were preincubated for 30 min, 25 nM Ub-Rho110 (Ubiquigent) was added to initiate the reaction, and the fluorescence intensity was recorded for 30 min on a PHERAstar FSX (λ_Ex = 485 nm, λ_Em = 520 nm) (BMG Labtech). Initial rates were plotted against compound concentration to determine IC₅₀. Data were processed using analysis tools from Dotmatics (https://www.dotmatics.com/). RapidFire MS coupled to an Agilent QTof 6530 mass spectrometer (30 (link)) was used to confirm USP30 complex formation with USP30_inh and assess cleavage of K6-linked di-Ub chains from USP30 in the presence and absence of the compound.

Ceramide and sphingomyelin concentrations were quantified by rapid resolution liquid chromatography/mass spectrometry. Short lipids were extracted from colon biopsies with C17-ceramide and C16-d31sphingomyelin as internal standards, after homogenization of colonic tissue. Subsequently, samples were analyzed by rapid-resolution liquid chromatography-MS/MS using a Q-TOF 6530 mass spectrometer (Agilent Technologies, Waldbronn, Germany) operating in the positive ESI mode. The subsequent quantification was performed using Mass Hunter Software, and the resulting sphingolipid quantities were normalized to the actual protein content of the homogenate.

Samples were analyzed by direct infusion on a QExactive mass spectrometer (Thermo Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were analyzed in both positive and negative ion modes with a resolution of R(m/z = 200) = 280,000 for MS and R(m/z = 200) = 17,500 for MSMS experiments, in a single acquisition. MSMS was triggered by an inclusion list encompassing corresponding MS mass ranges scanned in 1 Da increments (Surma et al., 2015 (link)). Both MS and MSMS data were combined to monitor CE, DAG, and TAG ions as ammonium adducts; PC, PC O-, as acetate adducts; and CL, PA, PE, PE O-, PG, PI, and PS as deprotonated anions. MS only was used to monitor LPA, LPE, LPE O-, LPI, and LPS as deprotonated anions; Cer, HexCer, SM, LPC, and LPC O- as acetate adducts. Additional Cer and SM analyses (University of Potsdam) were carried out with a 1260 Infinity LC system coupled to a QTOF 6530 mass spectrometer (Agilent Technologies) operating in the positive electrospray ionization mode (ESI+). The precursor ions of Cer or SM species (differing in their fatty acid chain lengths) were cleaved into the fragment ions m/z 264.270 or m/z 184.074, respectively (Kachler et al., 2017 (link)).