Metabolomics of Aldh7a1-/- mice treated with lysine alpha-oxidase from Trichoderma viride
Description
Metabolite analysis of brain, liver, and plasma isolated from Aldh7a1-/- (n=3/each) fed diets consisting of 0.9% w/w lysine and 18 ppm pyridoxine administered with PBS or 150 U/kg lysine alpha-oxidase (LOX) from Trichoderma viride. Tissues were cryo-homogenized using a liquid nitrogen cooled mortar and pestle into a fine powder and approximately 25-50 mg of each tissue was transferred to a pre-weighed homogenization tube to which ~20 2.3 mm zirconia beads and 1 mL of 80% ice cold methanol were added. Tissues were homogenized using a bead mill and cleared by centrifugation at 4C, where each of the 3-4 cycles consisted of a 60 second homogenization at 6 m/s followed by a 30 second pause. A volume equivalent to 25 mg of tissue extract was transferred to a new tube, and 250 ul of 80% methanol containing 1 ul of stable isotope-labeled internal amino acid standard mix was added. Metabolites were concentrated using a SpeedVac until dry. For plasma metabolite analysis, 10 µl of plasma was added to 250 µl of 80% methanol containing 1 µl of stable isotope-labeled internal amino acid standard mix, vortexed at 4C for 10 minutes, and centrifuged. 0.9 ml of supernatant was transferred to a new tube and concentrated using a SpeedVac until dry. Metabolites were reconstituted into 25-50 µl of water, vortexed, centrifuged, and transferred to vials for analysis by LCMS. LCMS was performed using a ZIC-pHILIC LC column coupled to a Vanquish LC and a flow gradient consisting of 10 mM ammonium carbonate in water and pure acetonitrile. The LC was coupled to an Exploris 240 mass spectrometer operated in a polarity switching data-dependent Top 5 mode. Full MS scan parameters for both positive and negative mode were set to 67-1000 m/z at a resolution of 120k and ddMS2 were collected at a resolution of 30k. Data files are labeled with brain, liver, and plasma followed by a number. The number represents a mouse. Samples 1-3 are from PBS-treated mice and samples 4-6 are from LOX-treated mice.