Metabolite extraction from adherent mammalian cells
Page last modified on: 2024, November 04
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:
- 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]
- Aspirate the medium completely.
- 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.
- 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)
- Incubate the plate at - 80 °C for 20 min. Remove cell plate and keep it on dry ice.
- Scrape the plates on dry ice with cell scraper.
- 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
- Add appropriate internal standards
- Vortex for 5 min at maximum speed and make sure that pellet disintegrates and mixed thoroughly with extraction solvent.
- 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.
- Centrifuge the tube at 14,000g for 10 min at 4–8 °C to pellet the cell debris.
- Transfer the metabolite-containing supernatant to a new 15-ml conical tube (or 1.5 ml eppendorf) on dry ice.
- 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)
- Spin the tubes at 14,000g for 10 min at 4–8 °C.
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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]
- SpeedVac/lyophilize or dry under nitrogen gas to a pellet using no heat.
- Submit dried sample in 1.5 ml eppendorf tube and can be stored at in dried ice.
- Blank control: prepare processed blank sample using same procedure but without biological sample (use water or buffer instead).
References
- 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