Cecal Metabolome Profiling in Mice

Cecal contents from 13 weeks old mice which stored at −80°C were weighed and completely homogenized in 500 µl methanol containing 50 µM methionine sulfone and camphor-10-sulfonic acid as internal standards. The homogenates were mixed with 500 µl chloroform and 200 µl Milli-Q water. Samples were centrifuged (2,300 g, 5 min, 4°C), and then the supernatant was centrifugally filtered using 5-kDa cut-off filters (Millipore, Bedford, MA) until all was filtered (9,100 g, 4°C). The filtrate was centrifugally concentrated in a vacuum evaporator, dissolved in Milli-Q water and analyzed by capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE-TOFMS).
CE-TOFMS analysis was performed by an Agilent CE system combined with a TOFMS (Agilent Technologies, Palo Alto, CA) as reported by Human Metabolome Technologies Inc. (HMT, Tsuruoka, Japan).^{(11 (link),12 (link))} Each metabolite was identified and quantified based on the peak information including m/z, migration time and peak area.