DRC Regional Metabolomics and Fluxomics Core - Resources

Resources like useful websites, reference papers, links to MAVEN, links to natural isotope correction software, compound lists.

Water-Soluble Metabolite Extraction of Serum

Serum (3 µL) is added to 90 µL of -80*C 100% methanol, vortexed, and put on dry ice for at least 5 min. This mixture is then centrifuged at 16,000 x g for 10 min at 4 °C. The supernatant is then transferred to tubes for LC-MS analysis.

Water-Soluble Metabolite Extraction of Tissue

Tissues clamped and frozen in liquid nitrogen are homogenized via Cryomill at cryogenic temperature (Retsch, Newtown, PA). Around 20 mg of tissue is then weighed and mixed with -20 °C 40:40:20 methanol:acetonitrile:water at a concentration of 25 mg/mL. Samples are then vortexed and centrifuged twice at 16,000 x g for 20 min at 4 °C before transferring the supernatant to LC-MS tubes for analysis.

Metabolite Measurement by LC-MS

A quadrupole-orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific, San Jose, CA) operating in negative mode is coupled to hydrophilic interaction chromatography (HILIC) via electrospray ionization. Scans are performed from m/z 70 to 1000 at 1 Hz and 140,000 resolution. LC separation is on an XBridge BEH Amide column (2.1 mm x 150 mm x 2.5 mm particle size, 130 A° pore size; Waters, Milford, MA) using a gradient of solvent A (20 mM ammonium acetate, 20 mM ammounium hydroxide in 95:5 water:acetonitrile, pH 9.45) and solvent B (acetonitrile). Flow rate was 150 µL/min. The LC gradient was: 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min,0% B; 23 min,0% B; 24 min, 85% B. Autosampler temperature was 5 °C, and injection volume was 5-10 µL for serum samples and 10-15 µL for tissue samples.

El-Maven (Elucidata) is used for analysis of LC-MS data.

Isotope labeling is corrected using AccuCor, available on GitHub.

Metabolite Extraction of Serum and Tissue For Lipidomics

Water-Soluble Metabolite Extraction of Cultured Cells

Knowns List (pdf)

Best Practices for Shipping Samples