Bindley Bioscience Center

High Throughput Metabolite Profiling

Global GCxGC/MS Profiling Analysis Procedure

Tissue Extraction:

1. Add 200 uL serum to an epi tube
2. Add 400 uL MeOH. Remain at room temperature for 1 hour, vortex every 20 minutes.
3. Place in -20 °C freezer for 30 minutes.
4. Remove from freezer. Spin down while cold, 14,000 RPM for 10 minutes. {proteins will pellet}
5. Remove the MeOH supernatant.
6. To the protein pellet, add 200 uL CHCl3. Vortex.
7. Spin down (13,000 g) for 10 minutes.
8. Remove the CHCl3 supernatant. {contains fatty acids}
9. Combine the MeOH supernatant and the CHCl3 supernatant in a fresh epi tube. Volume is about 600 uL.
10. SpeedVac to dryness, about 90 minutes. Can store -80 °C.

TBDMS Sample Derivatization:

1. Add 50 uL anhydrous pyridine via syringe.
2. Remove 20 uL and add to a fresh epi. Freeze the other 30 uL aliquot as a back-up.
3. To the 20 uL of anhydrous pyridine solution, add 20 uL MTBSTFA.
4. Heat at 60°C for 1 hour.
5. Transfer contents to a glass GC micro autosampler vial.
6. Submit for chromatography.

Global LC/MS Profiling Analysis Procedure

Tissue Extraction:

1. Add 50 uL of plasma to an epi tube.
2. Add 150 uL of cold MeOH.
3. Vortex for 1 minute.
4. Place in -20 °C freezer for 30 minutes.
5. Remove from freezer. Spin down while cold, 14,000 RPM for 10 minutes. {proteins will pellet}
6. Remove the MeOH supernatant.
7. SpeedVac to dryness, about 90 minutes. Can store -80 °C.
8. Resuspend in 50 uL of 20% MeOH/80% aqueous mobile phase.
9. Submit for chromatography.