| Description |
The analytical instrument used in this study was a Dionex U3000 UHPLC (ultra-high-performance liquid chromatography system) coupled with a QE Plus high-resolution mass spectrometer. For metabolite extraction, 20 μL of 2-chloro-l-phenylalanine (0.3 mg/mL) dissolved in methanol, serving as the internal standard, and 1 mL of a methanol:water mixture (4: 1=v: v) were added to each sample. The resulting mixture was then transferred to a 4 mL glass vial. Subsequently, 200 μL of trichloromethane were subsequently added to each aliquot to disperse the sample using a pipette. The cells were disrupted using an ultrasonic homogenizer at 500 watts for 6 min.All of the mixtures from each sample were transferred to 1.5 mL Eppendorf tubes and extracted by ultrasonication for 20 min in an ice water bath. The resulting extract was centrifuged at 13,000 rpm and 4 for 10 min. Thereafter, 1 mL of the supernatant was then dried in a freeze concentration centrifugal dryer in a glass vial. A volume of 200 μL of a methanol-water mixture (1:4, vol/vol) was added to each sample, which were then vortexed for 30 s and placed at 4°C for 2 min.Subsequently, the samples were centrifuged at 13,000 rpm and 4°C for 15 min. The supernatants (150 μL) from each tube were collected using crystal syringes, filtered through 0.22 μm microfilters, and transferred to LC vials. The vials were stored at -80°C until LC-MS analysis.The acquired LC-MS raw data were analyzed using the Progqenesis QI software (Waters Corporation, Milford, USA). Metabolites were identified using the Progenesis QI Data Processing Software (Waters Corporation, Milford, USA), which employs a combination of public databases, including http://www.hmdb.ca/ and http://www.lipidmaps.org/, and self-built databases. All of the aforementioned analyses were conducted by OE Biotech (Shanghai, China) |
