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Metabolite extraction from adherent mammalian cells

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Protocol/MCF/SamplePrep/01: Metabolite extraction from adherent mammalian cells (Yuan et al., 2012)

Aim: Aqueous metabolite extraction from mammalian cells or microbial samples for LC-MS analysis

Sample required: Cells required: Cells For cultured cells, use the equivalent of 2–3 million cells. For other sample types a minimum mass of 20 mg is required.

Materials: MS grade Methanol (with suitable IS mix 100 ng), MilliQ Water, with suitable internal standards mix 100 ng

Note: Keep diluents in chilled condition (0-4oC)
Sample preparation and metabolite extraction:

  1. Take 250μl spent growth medium and place it in Eppendorf tubes which already contain 750μl very cold (-80oC) HPLC-grade methanol. (Keep it in case you wish to analyze extracellular metabolites). [optional]
  2. Aspirate the medium completely.
  3. Pour 10ml (adjust volume as per the size of dish/ plate) of 10 mM Ammonium acetate (all over the petri dish, wash cells carefully and gently and then discard the washing solution.
  4. Put the plates on dry ice and add 4 ml of 80% (vol/vol) methanol (or Methanol:acetonitrile:water; 4:4:2) (cooled to - 80°C or on dry ice or liquid nitrogen). (adjust volume as per the size of dish/ plate)
  5. Incubate the plate at - 80 °C for 20 min. Remove cell plate and keep it on dry ice.
  6. Scrape the plates on dry ice with cell scraper.
  7. Transfer the cell lysate/methanol mixture to a 15 ml conical tube (or 1.5 ml / 5 ml eppendorf depending on volumes) on dry ice. If you notice residual cells on the plate, introduce additional washing steps to increase the yield and reduce inter-sample variability
  8. Add appropriate internal standards
  9. Vortex for 5 min at maximum speed and make sure that pellet disintegrates and mixed thoroughly with extraction solvent.
  10. Sample lysis and homogenization (discuss details and alternatives with MCF member). Simplest procedure: place the samples in an ultrasonication bath and sonicate the sample tube for 5 min (Note: Put some ice in sonicator water bath to avoid heating of sample during sonication; rotate/change position of samples during sonication as the energy is not homogeneously distributed in most ultrasonication baths) and vortex briefly after sonication.
  11. Centrifuge the tube at 14,000g for 10 min at 4–8 °C to pellet the cell debris.
  12. Transfer the metabolite-containing supernatant to a new 15-ml conical tube (or 1.5 ml eppendorf) on dry ice.
  13. Optional re-extraction step: Add 500 μl 80% (vol/vol) methanol (- 80°C) to the pellet and resuspend in a 1.5 ml tube and vortex for 1 min. ○ Resuspending the pellet may be difficult and may require a combination ofvortexing and pipetting up and down (or short period (5 sec) of ultrasonication)
  14. Spin the tubes at 14,000g for 10 min at 4–8 °C.

  15. Transfer the supernatant to a tube on dry ice (from Step 13).Divide and transfer 4.5 ml of total extraction buffer into three 1.5-ml microcentrifuge tubes (1.5 ml in each tube). [This step is optional, extracts can be dried directly]

  16. SpeedVac/lyophilize or dry under nitrogen gas to a pellet using no heat.
  17. Submit dried sample in 1.5 ml eppendorf tube and can be stored at in dried ice.
  18. Blank control: prepare processed blank sample using same procedure but without biological sample (use water or buffer instead).

References

  1. Yuan, M., Breitkopf, S. B., Yang, X., & Asara, J. M. (2012). A positive/negative ion–switching, targeted mass spectrometry–based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nature Protocols, 7(5), 872–881. https://doi.org/10.1038/nprot.2012.024