Untargeted metabolomics
About 10 mg of murine lungs were extracted by 80% methanol water to be
further analyzed by a quadrupole orbitrap mass spectrometer (Orbitrap
Exploris 480; Thermo Fisher Scientific) that is coupled to a Vanquish
UHPLC system (Thermo Fisher Scientific) with
electrospray ionization (ESI) or
atmospheric pressure chemical
ionization (APCI) and scan range m z-1 from 70 to
1000, with a 120,000 resolution. LC separation was performed on an
XBridge BEH Amide column (2.1×150 mm, 2.5 μm particle size; Waters)
using a gradient of solvent A (95:5 water: acetonitrile with 20 mM of
ammonium acetate and 20 mM of ammonium hydroxide, pH 9.45) and solvent B
(acetonitrile).The flow rate was 150 μL min-1. The LC
gradient was: 0 min, 90% B; 2 min, 90% B; 3 min, 75%; 7 min, 75% B;
8 min, 70% B, 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min,
25% B; 14 min, 25% B; 16 min, 0% B, 21 min, 0% B; 21 min, 90% B;
and 25 min, 90% B. Injection volume was 5-10 μL and autosampler
temperature was set at 4℃. All metabolite classifications were based on
the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). KEGG pathway
enrichment analysis was performed using MetaAnalyst
(https://www.metaboanalyst.ca/).
Heatmap, dot plot, Venn diagram and multi-dot were plotted by
bioinformatics (https://www.bioinformatics.com.cn) , the
same website mentioned above.