| 描述信息 |
The gas chromatography mass spectrometry system was applied for metabolomic analysis. Samples were treated with methanol H2O and then transferred to a glass vial. Chloroform was added to each aliquot, and the samples were broken up by ultrasonic homogenizer. The extract was centrifuged at 13000 rpm for 10 min at 4°C to obtain the supernatant, which was subsequently dried in a freeze concentration centrifugal dryer. The derivatized samples were analyzed on an Agilent 7890B GS system, coupled with an Agilent 5977A MSD system (Agilent Technologies Inc., USA). Quality control (QC) samples were applied to monitor the stability and repeatability of instrument analysis.For metabolomics data analysis, the obtained GC/MS raw data were converted into data format files. Then data were imported into MS-DIAL software for peak detection, peak identification, MS2Dec deconvolution, characterization, peak alignment, wave filtering, and missing value interpolation. Metabolite characterization was based on LUG database. A data matrix was derived. The three-dimensional matrix includes: sample information, the name of the peak of each substance, retention time, retention index, mass-to-charge ratio, and signal intensity. In each sample, all peak signal intensities were segmented and normalized according to the internal standards with relative standard deviation (RSD) < 0.1 after screening.After the data was normalized, redundancy removal and peak merging were conducted to obtain the data matrix. Principle component analysis (PCA) was conducted to observe the overall distribution among the samples and the stability of the whole analysis process. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and partial least squares-discriminant analysis (PLS-DA) were utilized to distinguish the metabolites that differ between groups.Variable importance of projection values obtained from the OPLS-DA model were used to demonstrate the overall contribution of each variable to group discrimination. A two-tailed Students t-test was further applied to verify the significance of metabolite differences between groups. |
