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.