Untargeted and targeted metabolomics

Metabolites were extracted using a liquid–liquid extraction (LLE) method and a mixture of methanol/chloroform/hexane (2/0.5/0.5, v/v/v). Further analyses were carried out using a Thermo Scientific LTQ Orbitrap™ Hybrid Ion Trap-Orbitrap mass spectrometer (MS) and a Waters 2795 HPLC separation module interfaced by an electrospray ionization (ESI) source. The metabolites were separated on a Scherzo SMC₁₈ column (150 mm × 4.6 mm; 3-µm particle size; Imtakt, Portland, OR). The mobile phase used for chromatographic separation was as follows: solvent A, 10 mM ammonium acetate:acetonitrile (40:60, v/v); solvent B, 2-propanol:acetonitrile (90:10, v/v). The optimized chromatographic gradient conditions were as follows: 0 min, 95% A; 2 min, 95% A; 22 min, 10% A; 27 min, 10% A; 28 min, 95% A; 35 min, 95% A; the flow rate was set at 0.5 ml/min. Data acquisition and analysis were conducted using Thermo Xcalibur^TM software. The data were collected separately in the positive and negative electrospray ionization modes, and the acquisition was performed in the centroid mode using the full-scan MS method. The general mass spectrometer operating conditions were as follows: Spray voltage: + 4.5 kV for the positive mode and −3.5 kV for the negative mode; source temperature: 350 °C; vaporizer temperature: 450 °C; sheath gas: 35 arbitrary units; auxiliary gas: 10 arbitrary units; sweep gas: 2 arbitrary units; mass scan range: m/z 50–1200 at 0.1 scan/s.

For MS/MS analysis, the mass parameters were as follows: isolation width: 2.0 (m/z); collision-induced dissociation (CID): 40%; resolution: 30,000; maxIT: 100 ms. An open source software package MZmine v.2.21 (http://mzmine.sourceforge.net/) was used for data processing. The RANSAC algorithm was applied to align the detected peaks in the different samples, generating an aligned peak list for Slc29a3^+/+ & Slc29a3^−/− samples. A database search using the Human Metabolome Database (HMDB) (http://www.hmdb.ca), KEGG pathway database (http://www.genome.jp/kegg/) & Lipid Metabolites and Pathway Strategy (Lipid Maps) (http://www.lipidmaps.org) was performed to establish peak identities for each m/z in the aligned peak list with a mass tolerance set to ± 10 ppm. Lipidomics analysis of the liver samples from Slc29a3^+/+ & Slc29a3^−/− was performed using the method described by Park et al.⁶⁴

Functional enrichment of the metabolome data with KEGG pathway annotations was performed using MBROLE2⁶⁵ (http://csbg.cnb.csic.es/mbrole2/). Integrated pathway analysis of transcriptome and metabolome data was performed using MetaboAnalyst^®3.0⁶⁶ by employing the KEGG pathway database. The integrated metabolomics and transcriptomics network was constructed based on the HMDB ID for metabolites and gene symbols for transcripts using the open source software GAM⁶⁷ (https://artyomovlab.wustl.edu/shiny/gam), ipath⁶⁸, and LRpath⁶⁹.