Nexera uhplc

Reduced and alkylated tryptic peptides (5 μL, corresponding to 5 μg extracted protein) were chromatographically separated on a Shimadzu Nexera UHPLC and analyzed on a 6500 QTRAP mass spectrometer (SCIEX) as described previously (Colgrave et al., 2014 (link)). Quantification was achieved using scheduled MRM scanning experiments using a 40 s detection window for each MRM transition and a 0.3 s cycle time. Peaks were integrated using MultiQuant v3.0 (SCIEX) wherein all three transitions were required to co-elute at the same retention time (RT, min) with a signal-to-noise (S/N) > 3 for detection and a S/N > 5 for quantification. Graphs were generated in Graphpad Prism v6.

PK studies were performed in male CD-1 mice between 18 and 25 g in weight. The test compound was injected intravenously (i.v.) into CD-1 mice at 0.5 mg/kg in formulation containing 5% DMA (dimethyl acetamide) 15% Solutol HS15 and 80% water. Animals were euthanized at predefined time points by CO₂ inhalation and blood was collected in K2-EDTA vacutainers by cardiac puncture. The vials were then centrifuged to collect plasma. The plasma samples were extracted using acetonitrile and then analyzed by LC-MS to determine the concentrations of Compound 1 and Compound 3. LC-MS was performed using a Luna Omega 1.6 μm Polar C18 (Phenomenex) column, a Shimadzu Nexera UHPLC, and a Sciex QTrap 5500 mass spectrometer. The PK parameters were calculated by non-compartmental analysis using Phoenix WinNonlin (Certara).

Reduced and alkylated tryptic peptides (5 μl) were chromatographically separated on a Kinetex C18 column (2.1 mm x 100 mm, Phenomenex) using a linear gradient of 5–45% acetonitrile (in 0.1% formic acid) over 10 min at a flow rate of 400 μl/min. The eluent from the Shimadzu Nexera UHPLC was directed to a QTRAP 6500 mass spectrometer (SCIEX) equipped with a TurboV ionization source operated in positive ion mode for data acquisition and analysis. The MS parameters were as follows: ion spray voltage, 5,500 V; curtain gas, 35; GS1, 35; GS2, 40; source temperature, 500°C; declustering potential, 70 V; and entrance potential, 10 V. Peptides were fragmented in the collision cell with nitrogen gas using rolling collision energy dependent on the size and charge on the size and charge of the precursor ion. Relative quantitation using scheduled multiple reaction monitoring (MRM) scanning experiments (MRM transition peptide information provided in Supplementary Table 3) with a 40 second detection window around the expected retention time (RT) and a 0.3 second cycle time. Data were acquired using Analyst v1.7 software. Peak areas of four MRM transitions were integrated using Skyline (MacLean, Bioinformatics 2010) wherein all transitions were required to co-elute with a signal-to-noise (S/N) > 3 and intensity >1,000 counts per second (cps) for detection.