Fecal Metabolites_original.XLSX

Fecal and serum samples were
maintained at -80◦C until metabolic profiling was performed. Global
non-targeted mass spectrometry metabolomics analysis was performed at
Metabolon, Inc (Metabolon, Inc, Durham, NC), a commercial supplier of metabolic
analysis, which has developed a platform that integrates chemical analysis (including
identification and relative quantification), data-reduction, and quality
assurance. To maximize compound detection and accuracy, 3 separate analytical
methods were utilized including ultra-high performance liquid chromatography-tandem
mass spectrometry (UHPLC-LC-MS) in both positive and negative ion modes and gas
chromatography/mass spectrometry (GC-MS) (40, 41). Following receipt, samples were inventoried,
accessioned into the Metabolon Laboratory Information Management System (LIMS),
assigned a unique identifier, and stored at -80◦C until analyzed.

Sample preparation was performed
by the automated Mircolab STAR system (Hamilton Company, Salt Lake City, UT,
USA). Extraction solvents (i.e.,
methanol containing recovery standards) were added to each sample. Extraction
was carried out by shaking using a Geno/Grinder 2000 (Glen Mills, Clifton, NJ)
followed by centrifugation. The resulting extract was divided into 5 fractions:
2 for analysis by 2 separate reverse phase (RP) LC/MS methods with positive ion
mode electrospray ionization (ESI), 1 for analysis by RP LC/MS with negative
ion mode ESI, 1 for GC/MC analysis, and 1 reserve aliquot. Samples were placed
on a TurboVap® (Zymark, Midland, Ontario, Canada) to remove organic solvent and
were stored overnight under nitrogen before preparation for analysis.

All UPLC-MS/MS methods
utilized a Waters Acquity UPLC (Walters,
Milford, MA, USA) and a Q-Exactive high resolution/accurate mass spectrometer
interfaced with heated electrospray ionization (HESI-II) source and Orbitrap
mass analyzer operated at 35,000 mass resolution (Thermo Scientific, Walthram,
MA, USA). Sample extracts were reconstituted in solvents compatible to each method
employed. The MS analysis alternated between MS and data-dependent MSn
scans using dynamic exclusion. The scan
range varied slighted between methods but covered mass to charge ratios of 70
to1000 m/z.
Raw data were archived
and extracted. For GC/MS, derivatized
samples were separated on a 5% phenyldimethyl silicone column with helium
carrier gas. Data were analyzed on a Thermo-Finnigan Trace DSQ MS (Thermo
Fisher) operated at a unit mass resolving power with electron impact
ionization.

Detection,
identification, integration, and clustering of all ion features into individual
compounds was performed using a library of conserved chemical standards using a
software developed by Metabolon (42). Authenticated
standards within the library contain retention time/index (RI), mass to charge
ratio (m/z), and chromatographic data
(including MS/MS spectral data). At present, 3,000 commercially available
purified standards are registered into the LIMS for distribution to both the LC
and GC platforms for determination of their analytical characteristics.
Compound abundance was quantified by calculation of area under the curve for the
quantification ion of the compound. Quality control measurements were used to individually
check all compounds.