Untargeted Metabolomics
Prior to analysis, aqueous extracts were resuspended in 100 μL of 2% acetonitrile in water. Chromatographic separation was performed on a reversed-phase ACQUITY UPLC HSS T3 1.8 μm column (i.d. 3.0 × 100 mm) (Waters) using an UPLC system (Agilent 1290 Infinity II; Agilent Technologies) as described previously (Tian et al., 2020). MS detection was performed using high-resolution time-of-flight (TOF) mass spectrometry (5600 Triple TOF Plus, Sciex) equipped with an ESI source (Yuan et al., 2012). Data were acquired in TOF full scan method with positive and negative ion modes, respectively. Information-dependent acquisition methods were used for MS/MS analyses of metabolome. The collision energy was set at 35 ± 15 eV. Metabolite identification was compared with standard references, HMDB (https://hmdb.ca/), METLIN (https://metlin.scripps.edu), and literature searches. A total of 45 isotopically-labeled internal standards (IS), purchased from Cambridge Isotope Laboratories, were spiked into the samples for metabolite quantitation, including L-Phenylalanine-d8, L-Tryptophan-d8, L-Isoleucine-d10, L-Asparagine-13C4, L-Methionine-d3, L-Valine-d8, L-Proline-d7, L-Alanine-d7, DL-Serine-d3, DL-Glutamic acid-d5, L-Aspartic acid-d3, L-Arginine-d7, L-Glutamine-d5, L-Lysine-d9, L-Histidine-d5, Taurine-d2, Betaine-d11, Urea-(13C,15N2), L-lactate-13C3, Trimethylamine N-oxide-d9, Choline-d13, Malic acid-d3, Citric acid-d4, Succinic acid-d4, Fumaric acid-d4, Hypoxanthine-d3, Xanthine-15N2, Thymidine (13C10,15N2), Inosine-15N4, Cytidine-13C5, Uridine-d2, Methylsuccinic acid-d6, Benzoic acid-d5, Creatine-d3, Creatinine-d3, Glutaric acid-d4, Glycine-d2, Kynurenic acid-d5, L-Citrulline-d4, L-Threonine-(13C4,15N), L-Tyrosine-d7, P-cresol sulfate-d7, Sarcosine-d3, Trans-4-hydroxy-L-proline-d3, Uric acid-(13C; 15N3). Metabolite levels were normalized according to the following rules (1) ISs were applied to correct peak areas of their corresponding metabolites; (2) When (1) was not feasible due to unavailability of commercial standards, peak areas were corrected with IS of metabolites of the same class, comparable peak intensities, and/or proximity in retention times; (3) Results from Step (2) were evaluated based on relative standard deviation (RSD) values of each metabolite before and after IS correction. Corrected peak areas were adopted if their corresponding RSDs were smaller than that of original areas in quality control samples. For simplicity, all metabolite levels were labeled as “intensity.”