UHPLC-OE-MS untargeted metabolomics:
Blood samples collected in serum separator tubes were subjected to centrifugation at 4°C and 1500×g for 10 minutes. The resulting serum was aliquoted and stored at -80°C until analysis. A 100 μL sample was combined with 400 μL of an extraction solution (MeOH: ACN = 1:1, including an isotopically-labelled IS mixture) in an EP tube. The mixture underwent vortexing for 30 seconds, followed by sonication for 10 minutes in an ice-water bath. Subsequently, it was incubated at -40°C for 1 hour to facilitate protein precipitation (PPT). After centrifugation at 12000 rpm (RCF = 13800 xg, R = 8.6 cm) for 15 minutes at 4°C, the resulting supernatant was transferred to a fresh glass vial for analysis. Additionally, a quality control (QC) sample was created by combining equal volumes of supernatants from all samples.
A UHPLC system (Vanquish, Thermo Fisher Scientific) coupled with an Orbitrap Exploris 120 mass spectrometer (Orbitrap MS, Thermo) and a Waters BEH Amide column (2.1 mm × 50 mm, 1.7 μm) was used for LC-MS/MS analyses. The mobile phase comprised a water-based solution (pH = 9.75) containing NH4OAc and NH4OH (A), as well as acetonitrile (B). The autosampler temperature was maintained at 4°C, and 2 μL injections were introduced. The mass spectrometer operated with Xcalibur software in information-dependent acquisition (IDA) mode. ESI source conditions included sheath gas flow rate = 50 Arb, auxiliary gas flow rate = 15 Arb, capillary temperature = 320°C, full MS resolution = 60000, MS/MS resolution = 30000, collision energy = 20/30/40 in NCE mode, and spray voltage at 3 kV (positive) or -3 kV (negative). The raw data obtained from the experiments were converted into mzXML format using ProteoWizard. Subsequently, an in-house program developed in R, incorporating XCMS, was utilized for peak detection, extraction, alignment, and integration processes. Metabolite annotation utilized the MS2 database (BiotreeDB), KEGG, and HMDB, with an annotation threshold set at 0.3.