Untargeted Metabolomics
Methods
Sample extraction. Samples were weighed at 5 mg and extracted with 250 µL of methanol and 250 µL of 10 mm ammonium acetate. Mixing and homogenization for 30 s at 1800 rpm was done using the Beadbeater. Samples were sonicated for 10-20 min at room temperature and centrifuged at 20,000 x g for 10 min at room temperature. 200 µL of supernatant was transferred to a clean Eppendorf tube for drying under nitrogen at 30°C. Samples were reconstituted with 50 µL of water with 0.1% formic acid, centrifuged, and transferred to clean LC vials.
LC-MS Analysis. Global metabolomics profiling was performed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 µm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. The flow rate was 350 µL/min with a column temperature of 25° C. 4 µL was injected for negative ions and 2 µL for positive ions.
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Results
Our laboratory received 4 Excel files with the results of the untargeted analyses. Two separate files for the pig and ding samples contained qualitative data with the name and molecular formula of the compound. The ChemSpider and MzCloud databases were used to identify the compounds of approximately 200 molecules. Two additional files of the positive and negative polar molecules contained quantitative data such as the retention time (RT), mass to charge ratio (M/Z), and peak height. While there were over 4000 negative molecules & 5000 positive molecules with RT and M/Z recorded, a very small percentage of molecules were identified with the name and molecular formula.
Statistical analyses on the quantitative data were performed using the SECIM Tools in Galaxy, a python platform. SECIM Tools are interfaced to be user friendly and provide a large selection of statistical procedures. There is a detailed user guide available to researchers as well as a recently published article (Kirpich, et al., 2018). Currently, our laboratory has performed the mass to charge ratio/RT analysis that provided data on matched and unmatched molecules between the pig and ding samples with positive and negative polarity. Three Venn diagrams were generated for matched and unmatched features, combinations, and multiples. The results for the matched and unmatched combinations are presented in Figs. 1-4 since these data provide the most comprehensive perspective.