I'm curious if anyone here has any experience in creating metabolite extracts, bacterial in my case but I think it wouldn't be too hard to modify other protocols maybe for mammals to suit my needs. I'm interested in getting the fullest spectrum of chemical species possible.

I have a few places to start but I'm wondering maybe someone reading this has more experience and can possibly suggest a couple decent protocols.

For metabolome analyses we disrupt ~10-30 mg cells in 80 % methanol using bead beating.

-incubation 15 min at 70° C

-centrifugation

-supernatant was evaporated

for GC/MS:

-Methoximation of carbonyl moieties:

50 μl of a 20 mg/ml solution of methoxylamine hydrochloride

in pyridine, incubation 37°C for 90 min (stirring)

-Protection of acidic protons:

50 μl N-methyl-N-[trimethylsilyl]trifluoroacetamide, incubation at 37°C for 30 min

This method most likely wont work for my purposes. The extract isn't being used for analysis but instead as a trigger for a biological reaction dependent on the presence of (a)certain metabolite(s). As I don't know the metabolite required, I'm attmepting an extraction that will yeild the widest array of chemical species possible.

 

Thus I'm attmepting to find a more physical versus chemical process.

 

1. My proposal is to pulverise the cells in liquid N2 with sand as fine as possible.

2. dissolve in a small amount of water and pass through a low molecular weight membrane so as to eliminate all cellular debris, proteins, complexes etc...

3. processing of the remaining low molecular weight chemical species by a free drying process, into a powder for subsequent use.

 

Basically I'm trying to generate an extract that will contain a metabolite capable of trigering a response in an experiment without knowing what this metabolite is in the first place. Thus I would like to maintain as many chemical species as possible.