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.

Contact

Address
Purdue University
Metabolite Profiling Facility (MPF)
Bindley Bioscience Center
1203 W. State St.
West Lafayette, IN 47907-2010

Bruce Cooper, Ph.D.

Bruce Cooper, Ph.D.
Facility Director

 

Amber Hopf Janasch

Amber Hopf Jannasch
Laboratory Manager