soil metabolome

For metabolite extraction, 100 mg rhizosphere soil was processed with methanol:water (3:1, v/v) containing internal standards, followed by grinding, ultrasonication, and centrifugation at 12,000 rpm for 15 min at 4 °C. Supernatants were collected for LC–MS/MS. The supernatant was transferred to a 2 mL injection vial for LC-MS/MS analysis. Equal volumes of supernatants from all samples were pooled to prepare a quality control (QC) sample. LC-MS/MS analysis was conducted on an UHPLC system (Vanquish, Thermo Fisher Scientific), with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) connected to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo) [76]. The mobile phase consisted of 5 mmol/L ammonium acetate and 5 mmol/L acetic acid aqueous solution (pH = 9.75) (A) and acetonitrile (B), with an autosampler temperature of 4°C and an injection volume of 2 μL. The Thermo Q Exactive HFX mass spectrometer was operated under control software (Xcalibur, Thermo), and both full MS and MS/MS data were collected. Detailed parameters: Sheath gas flow rate: 30 Arb, Aux gas flow rate: 10 Arb, Capillary temperature: 350°C, Full MS resolution: 120,000, MS/MS resolution: 7,500, Collision energy: 10/30/60 in NCE mode, Spray Voltage: 4.0 kV (positive) or -3.8 kV (negative).The metaboliteraw data were processed to remove single-peak noise and filtered based on the relative standard deviation (RSD). Peaks with more than 50% missing values in any single group or across all groups were excluded. Missing values were imputed using the minimum half method, and the data were normalized using internal standards.Metabolite identification was performed by matching accurate mass (MS1) and MS/MS spectra against reference libraries. The confidence of each annotation was assessed according to the Metabolomics Standards Initiative (MSI) levels. Features with MS/MS spectral matches to experimental or in silico libraries [platform levels B(i), B(ii) and B(iii)] were assigned to MSI Level 2 (putatively annotated compounds). Features matched only by accurate mass (platform level D) were assigned to MSI Level 3 (putative compound class). No identifications reached MSI Level 1 (confirmed structure), as this requires validation with authentic chemical standards for both retention time and MS/MS spectrum under identical analytical conditions. The MS2 match score (ranging from 0 to 1) was retained as an additional measure of spectral similarity..