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Lipid and fatty acid extraction protocol from biological samples

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Protocol/MCF/SamplePrep/02: Lipid and fatty acid extraction protocol from biological samples (Bird et al., 2011)

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

Sample required: Cells required: For cultured cells, use the equivalent of 2–3 million cells or 20-30 mg of microbial mass

Materials: MS grade Methanol, Acetonitrile (ACN), Chloroform, Isopropanol (IPA), MilliQ Water, suitable internal standards mix Note: Keep diluents in chilled condition (0-4oC)

Procedure for Lipid extraction: MeOH: Chloroform extraction (Caution: use only glass vials, syringe. No plastic items should be in contact with Chloroform)

  1. Add 200 µL of cold methanol (containing internal standards mix) for 106 cells or 25 mg microbial culture (in 2 mL glass vial)
  2. Vortex and mix thoroughly for protein precipitation
  3. Add 500 µL of chloroform (with glass syringe) and vortex and keep at cold for 10 min
  4. Add 200 µL water for phase separation
  5. Vortex and keep at cold for 10 min
  6. Centrifuge at 600 rpm for 5 min (using 15- or 50-mL falcon tubes inserted with glass vials)
  7. Carefully remove bottom Chloroform layer of 300 µL using syringe and transfer to new amber colour glass vial (with syringe)
  8. Keep it at speedvac for 20 min at RT or dry under nitrogen gas stream
  9. Dried samples can be stored or shipped in dry ice for analysis.
  10. Reconstitute is dried sample in IPA:MeOH (1:1)
  11. Blank control: prepare processed blank samples using the same procedure but without biological sample (use water or buffer instead).

Note: Method of measurement of cellular/microbial mass should be established with biologist/ microbiologist depending on experimental design. The method can be biomass weight or OD measurements which can be further used for normalization of lipid levels. Similar decision should be made for quenching procedures

Specific instructions for homogenization of frozen tissues (eg; liver) prior to lipid extraction 1) Starting with frozen tissue*, grind the tissue to a powder while frozen using a hand-held ceramic mortar and pestle. The mortar and pestle (any other apparatus eg; tweezers) should be liquid nitrogen cooled before and should be kept cold throughout the process. A CryoMill can also be used for the homogenization of the tissue.

2) Weigh the powder into a tared eppendorf tube. Keep track of the tissue weights as you need to correct for tissue weight (normalize) when resuspending the samples prior to LC-MS analysis

3) Extract lipids using the method above Protocol/MCF/SamplePrep/02

4) Re-extraction: After the initial extraction, perform a second extraction step of the tissue with 2:1 Chloroform: Methanol (~ 700μL) and combine the bottom layer with the layer from step 3.

5) Dry the bottom layer under a nitrogen stream.

*It is difficult to process tissue samples at weights below 10 mg, so aim for at least 10 mg of tissue to start with

References

  1. Bird, S. S., Marur, V. R., Sniatynski, M. J., Greenberg, H. K., & Kristal, B. S. (2011). Serum lipidomics profiling using LC–MS and high-energy collisional dissociation fragmentation: Focus on triglyceride detection and characterization. Analytical Chemistry, 83(17), 6648–6657. https://doi.org/10.1021/ac201195d