Masterview 1

The MS-DIAL platform was used for small molecule identification [80 (link)]. High-resolution Human Metabolome Database (HMDB) version 4.0 was used as a search space. For HMDB, relative intensities per component fragments SPLASH (SPectraL hASH; database-independent identifier) were filtered and stratified depending on their relative intensity. The latter with relative intensity greater than 75% were considered the main fragment (base peak), while fragments with relative intensities lower than 5% were excluded. Fragments >5% and <75% were considered as secondary fragments. Manual validation was performed to confirm ideal alignment using PeakView 2.2 with MasterView 1.1package (AB SCIEX) to give more credence to the identified molecules. The mass shift was 10 ppm, and the sample’s signal was at least five times higher than the blank. The identified small molecules were used for subsequent statistical analysis and biological evaluation.

MS-DIAL 3.70 open-source software (Tsugawa et al. 2015 (link)) was used for non-targeting, small molecule comprehensive analysis of the sample. According to the acquisition mode, ReSpect negative (1573 records) databases were used as reference databases. The search parameters were set as MS1 and MS2 mass tolerance: 0.01 Da and 0.05 Da for data collection, for peak detection; minimum peak height: 100 amplitude, mass slice width: 0.05 Da, smoothing level: 2 scans, minimum peak width: 6 scans, for identification MS1 and MS2 tolerance: 0.2 Da/each, for alignment; retention time tolerance: 0.05 min and MS1 tolerance: 0.25 Da. The MS-DIAL output was used to run again on PeakView 2.2 with the MasterView 1.1 package (AB SCIEX) for feature (peaks) confirmation from Total Ion Chromatogram (TIC) based on the criteria; aligned features having signal-to-noise ratio greater than 5 and intensities of the sample: blank greater than 5.

MS-DIAL 3.70 open-source software [26 (link)] was used for non-targeting, small molecule comprehensive analysis of the sample. According to the acquisition mode, ReSpect positive (2737 records) or ReSpect negative (1573 records) databases were used as reference databases. The search parameters were set as MS1 and MS2 mass tolerance: 0.01 Da and 0.05 Da for data collection, for peak detection; minimum peak height: 100 amplitude, mass slice width: 0.05 Da, smoothing level: 2 scans, minimum peak width: 6 scans, for identification MS1 and MS2 tolerance: 0.2Da/each, for alignment; retention time tolerance: 0.05 min and MS1 tolerance: 0.25 Da. The MS-DIAL output was used to run again on PeakView 2.2 with the MasterView 1.1 package (AB SCIEX) for feature (peaks) confirmation from Total Ion Chromatogram (TIC) based on the criteria; aligned features having Signal-to-Noise ratio greater than 5 and intensities of the sample: blank greater than 5.

PeakView 2.2, MasterView 1.1 package (AB SCIEX), and MultiQuant Software (AB SCIEX) were used to detect parents and fragment ions. Precursor ion XIC signal to noise ratio (S/N) > 3, sample: blank > 5 with a precursor mass tolerance of 10 ppm were applied as search parameters. High-resolution Human Metabolome database containing 114,100 metabolites (HMDB; downloaded March 2021) was used as a search space. For HMDB, fragments relative intensities per component (SPLASH) were filtered and stratified depending on their relative intensity. Samples were normalized using total ion chromatogram (TIC). Small molecules abundance data files were aggregated into one file using an in-house software tool and subjected to statistical analysis.