Metabolomic Profiling by LC-MS/MS

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed using an Ultra High Performance Liquid Chromatography (UHPLC) system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo) (Xue et al., 2021 (link)). The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra in information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur, Thermo). Metabolites were detected and analyzed in both positive- and negative-ion conditions to obtain the total ion current of the metabolites. After raw data were filtered and denoised, the final data set containing the peak number, sample name and normalized peak area information was imported into the SIMCA16.0.2 software package (Sartorius Stedim Data Analytics AB, Umea, Sweden) for multivariate analysis. Data were scaled and logarithmically transformed to minimize the impact of noise and high variance of the variables. Principal component analysis (PCA) was performed to visualize the distribution and grouping of the samples. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was performed in Metaboanalyst 5.0.