Q exactive combined quadrupole orbitrap mass spectrometer

Nanoliter high-performance liquid chromatography UltiMate 3000 RSLCnano (Thermo Fisher Scientific Company) was used in this study.
Q Exactive-combined quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific Company) was used in this study.
The chemicals and reagents used in this study were purchased from Sigma-Aldrich, including the standard protein product with the purity of 85% α-lactalbumin, 90% β-lactoglobulin, 70% α-casein, 98% β-casein, and alkaline bovine trypsin (Sigma, ≥10,000 N-benzoyl-l-arginine-ethyl ester, BAEE). The polyethersulfone fiber membrane was purchased from Agela and Phenomenex (Article No. AS051320-19). Mass spectrometry analysis software: Thermo Proteome Discoverer (version 1.4) from Thermo Fisher Scientific Company; Xcalibur (Thermo Fisher Scientific Company) ingredient list for desalting whey powder. A commercial infant formula powder has been used. Desalted whey powder from the model D90 was used (protein 14.01%, lactose 83.69%, and moisture 1%, Fonterra Company, New Zealand).

Liver microsomes were used to investigate the metabolism clearance of 7a. In addition, 5 µL of rat liver microsome (20 mg/mL), 2 µL of 7a or onvansertib (100 µM), and 173 µL of PBS buffer (0.1 M, pH 7.4) were added to 1.5 mL Eppendorf tubes. After pre-incubation for 10 min at 37 °C, the reaction was initiated by adding an NADPH-generating system (20 µL) into the microsomal suspension. A system without NADPH was set as a negative control. After incubations for 10 min, 30 min, or 60 min, 400 µL of ice acetonitrile was added to quench the reaction. The supernatants were injected into Q Exactive ™ combined quadrupole Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) for metabolite profiling and identification. The concentration of the compound to be measured at 0 min was defined as 100%, and the concentration at other times was converted into a residual percentage.