Tumor tissue
The snap-frozen liver and kidney tissues were lyophilized and
homogenized into powder. Approximately 10 mg of homogenized tissue was
transferred to a 2 mL Eppendorf tube, and 400 µL of methanol was added.
The mixture was then sonicated for 2 min and 1 mL of MTBE (methyl
tert-butyl ether) was added followed by 1 h of shaking in a shaking
water bath at room temperature. Separation was induced by the addition
of 250 µL of water followed by room temperature incubation for 10 min.
After 15 min of centrifugation at 14,000 rpm at 4 °C, the supernatant
was separated (upper organic layer and lower polar layer). For the
metabolomics study, 200 µL of supernatant was taken and evaporated to
dryness under a nitrogen stream at 37 °C. The dried residue was then
reconstituted in 100 µL of 80% methanol, and 50 µL was loaded into the
UHPLC-Orbitrap-MS system for analysis. For the tissue lipidomics study,
the same volume of supernatant was taken, processed following the same
method and finally reconstituted in 100 µL of chloroform/methanol (2:1).
In order to normalize tissue metabolomics and lipidomics data, the
protein concentration of each sample was quantified. Each sample was
diluted with distilled water, and the protein concentration was measured
by Nano-MD (SINCO, Korea) using 10 µL of sample. For both the tissue
metabolomics and lipidomics analyses, QC samples were made by taking
identical volumes from each sample and running the samples following a
sequence similar to that of plasma analysis.