Procedure: Liver metabolic profiling

Liver samples are analyzed at Metabolon (Durham NC); profiling complete in 6 days. The non-targeted metabolic profiling instrumentation employed combines three independent platforms: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS2) optimized for basic species, UHPLC/MS/MS2 optimized for acidic species, and gas chromatography/mass spectrometry (GC/MS). Three types of controls are analyzed in concert: aliquots of a pooled sample derived from a portion of all experimental samples (technical replicates), extracted water samples serve as process blanks, and a cocktail of standards spiked into every analyzed sample allow instrument performance monitoring. Experimental samples and controls are randomized across platform run days. Data are corrected by registering the median of each run-day to one followed by normalizing each data point proportionately.

1. Mice are fasted overnight and euthanized; liver harvested between 1000-1200h and flash frozen in liquid nitrogen.
2. Samples are processed as described (Evans et al., 2009; Ohta et al., 2009).
3. For each sample, 100 µL is used for analysis.
4. Using an automated liquid handler, protein is precipitated from the tissue-water homogenate with methanol containing four standards to monitor extraction efficiency.
5. Resulting supernatant is split into equal aliquots for analysis on the three platforms.
6. Aliquots are dried under nitrogen and vacuum-desiccated. Samples are reconstituted in 50 µL 0.1% formic acid in water (acidic conditions) or in 50 µL 6.5 mM ammonium bicarbonate in water (basic conditions) for the two UHPLC/MS/MS2 analyses or derivatized to a final volume of 50 µL for GC/MS analysis using equal parts bistrimethyl-silyl-trifluoroacetamide and solvent mixture acetonitrile:dichloromethane:cyclohxsane (5:4:1) with 5% triethylamine at 60°C for 1h.
7. For UHPLC/MS/MS2 analysis, aliquots are separated using ultrahigh performance liquid chromatography and LTQ mass spectrometer (consisting of an electrospray ionization source and linear ion-trap mass analyzer. The MS instrument scans 99-1000 m/z and alternates between MS and MS2 scans using dynamic exclusion with approximately 6 scans per second.
8. Derivatized samples for GC/MS are separated on a 5% phenyldimethyl silicone column with helium as the carrier gas and a temperature ramp from 60°C to 340°C and analyzed on a Thermo-Finnigan Trace DSQ MS operated at unit mass resolving power with electron impact ionization and a 50-750 atomic mass unit scan range.
9. Metabolites are identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra; curated by visual inspection for quality control using software developed at Metabolon.
10. False positives are minimized by filtering out metabolites that exhibit significant numbers of missing values (>20%).