Lcms 8045

The compounds targeted by the molecular networking strategy were quantified by using a UHPLC (Nexera X2; Shimadzu, Kyoto, Japan) coupled to a triple quadrupole mass spectrometer (LCMS-8045; Shimadzu, Kyoto, Japan) with an ESI source operated in positive ion mode. The conditions of chromatographic separation were the same as described in Section 3.3. The identity of the targeted compounds (evodiamine, dehydroevodiamine, and schinifoline) was confirmed by comparing the retention time and mass spectrum to their respective authentic standards. The contents of the three targeted compounds in the sample were calculated based on the calibration curves of external standards detected in multiple reaction monitoring (MRM) mode, and the MRM transitions were set as follows: evodiamine, m/z 304.1 → 134.1; dehydroevodiamine, m/z 302.1 → 286.1; and schinifoline, m/z 258.2 → 173.1. The method validation results are summarized in Tables S5–S8.

LC/MS 8045 (Shimadzu, Kyoto, Japan) consist of a Prominence-I LC-2030C 3D Plus LC unit and an ESI-TQ-MS detector. The apparatus was equipped with a Kinetex 2.6 µm C18 100A (100 × 3.0 mm) column with a Security Guard ULTRA 3 mm (Phenomenex, Torrance, CA, USA). For separation, 0.1% aqueous formic acid (A) and methanol (B) were used as mobile phases with a flow rate of 0.35 mL·min⁻¹ at 35 °C, with these gradient conditions: 10% to 20% B in 0–5 min; from 20% to 60% B in 5–10 min; from 60% to 10% B in 10–13 min and 10% B up to 17 min.
Identification was performed based on the optimised MRM mode (Table 2) according to polyphenol standards as a mixture (MetaSci, Toronto, ON, Canada) while quantification was carried out based on the external standard method.

Metabolites analysis including Methionine, Homocysteine, SAM, SAH, total Cysteine, Cystathionine, total Glutathione, Choline, Betaine, Glycine, Serine, methyl-THF and formyl-THF were realized by LCMS (LCMS 8045, Shimadzu, Kyoto, Japan) on a Kinetex column (Kinetex 00D-4462-EO). These measurements were performed as detailed in the Supplemental Methods section.

The S. quadricauda cells were collected by centrifugation at 8000 rpm for 10 min at room temperature. Metabolites were extracted from control or different time point samples from two independent biological replicate by homogenizing with liquid nitrogen in a prechilled sterile mortar and pestle. The samples then suspended with a mixture of 1 ml of methanol: water (80:20). Subsequently, the supernatant was collected by centrifugation at 8000 rpm for 10 minutes at 4°C and the extracted metabolites were stored at −20°C for LC-MS analysis. The mobile phase used for LC-MS is a mixture of triethylamine (A, 60%) and methanol (B, 40%) containing 0.1% formic acid adjusted to pH 4.2 and separated through a 1.9 μM C18 Shimadzu shim pack GISS column (Dimension 2.1 mm × 150 mm). The column temperature was maintained at 4°C and the temperature of the drying gas in the ionization source was 300°C. The gas flow was 10 l/min and the capillary voltage was 4 kV and the detection was using electrospray ionization (ESI)-MS. The LC-MS 8045 (Shimadzu, Japan) chromatogram was analyzed and the results were plotted by a heat map. The mean values of two experimental results were calculated and the data were used for the heat map generation (Supplementary Table 1). The heat map was generated using heat mapper (an online tool to interpret the metabolomic analysis) (Babicki et al., 2016 (link)).