Hi,
I plan to do metabolic profiling of cancer cells. For that, I am supposed to perform the following steps
1. Cell lysis
2. Cyroextraction using methanol and water
3. Vaccum evaporation and Freeze drying
4. LC/MS MS analytics of metabolites
5. Metabolomic analysis of generated data
My question is how to perform cyroextraction using methanol and water. Which grade methanol and water to be used. Also in what form should cell lysates be used before extraction begins?
Dear Pratik you can read the following paper
Small Molecule Metabolite Extraction Strategy for Improving LC/MS Detection of Cancer Cell Metabolome
Kathryn D. Sheikh, Shefali Khanna, Stephen W. Byers, Albert J. Fornace, Jr., and Amrita K. Cheema*
Hello,
A good platform to ask "metabolomics" oriented questions would be the Metabolomics Society Forum, intended for such questions and the answers are very focused and quick!
Try here: http://www.metabolomics-forum.com/
Before any start a good starting point would be:
Please consult this excellent review paper which lays down some guidelines which addresses all your questions! :)
Experimental design and reporting standards for metabolomics studies of mammalian cell lines: https://www.ncbi.nlm.nih.gov/pubmed/28669031 esp. tables 1 and 2.
Some other suggestions you would find here:
https://www.researchgate.net/post/Requesting_Adherent_cells_preservation_method_prior_to_Metabolite_extraction
Unsure why you have already "choosen" methanol: water ? Do you know which metabolites to target? If not then worth reading here:
Fiehn, O., Wohlgemuth, G., Scholz, M., Kind, T., Lee, D.Y., Lu, Y., Moon, S. and Nikolau, B., 2008. Quality control for plant metabolomics: reporting MSI‐compliant studies. The Plant Journal, 53(4), pp.691-704. ; where you can get idea on more better/ robust solvents to extract. A good one would be isopropanol: acetonitrile: water [3:3:2] unless you are targeting chemicals which are exclusively methanol: water specific?
Cold extraction solvents are good enough.
For cell lysis you can use any of those methods discussed in the Tengrove et al. paper [Experimental design and reporting standards for metabolomics studies of mammalian cell lines] which are specific for cell lines.
Also, depends whether you are using targeted or non-targeted LC-MS/MS platform? Whether you are aware of a list of chemicals (metabolites), then you are limited with the choice of solvents, vs. if you want to track a lot/ everything, you are free to try many and see what yields you best.....
Thanks,
Biswa
@ Biswada: Thank you so much BIswada! I understand that you already have so much experience in solvent extraction. Can I explain it to you directly here or your email. Mine is [email protected]
@ BIswada: I am working on glycoysis, PPP, nucleotide synthesis metabolism, glycine, serine synthesis pathways. The main metabolic pathways in cancer cells.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction