Hi,
If you are an expert in GC-MS metabolomics I need some help here!
I am doing a metabolomic analysis of a pure bacterial culture using derivatization with MTSFA using Fiehn´s protocol.
I am not very clear about how to prepare the cells for extraction and derivatization. I have a nutritive broth with a high cell count, then I have to somehow "clean it" so I can make the metabolite chemical extraction? or how do you account for the contents of the media on the sample?
In the protocol, it says I need to have a blank with only the media both treated as the samples. But is it not better to clean the sample first somehow?. I am confused.
Thanks,
Dear Lili Hernandez,
Firstly, I don't know about Fiehn's protocol. But, I can tell you some points which may be helpful to you, as I also derivatized my bacterial and cyanobacterial samples using MSFTA. As per my knowledge no need to have uninoculated media as treatment as it doesn't give any result.
You should be clear about your objective that whether you wanna do metabolite profiling of bacterial culture in one stage or different growth periods (namely 4h, 8h, 12h, 16h and so on). I recommend you to do time course as it will give you good results.
Coming onto your doubt, you can harvest the cells by centrifugation, followed by washing the cells with distilled water twice or thrice to remove the media traces. Then, weigh a known quantity of culture (20 mg for instance), grind it which liquid nitrogen, and follow the procedure. Make sure all the samples you have used should have same weight and internal standard should be there if you are doing untargeted GC/MS. If you are doing targeted GC/MS analyses then individual standards should be prepared...
Whatever you do scientifically is fine, but mention it in your paper or dissertation.
All the Best...
I hope this might help you...
Cleaning the cells means centrifuging the cells and washing them with a simple buffer system (e.g., phosphate-buffered saline). The less components in the wash buffer the better. The problem with this, however, is that the metabolic composition of the cell changes pretty quickly and you are effectively stressing the cells by doing this. You also want to avoid heat or cold shocks so the buffer should be at the same temp as the growth media. Snap freezing the pellet in liquid N2 or dry ice after the wash helps preserve the metabolites.
The issue with the derivatization will be what in the buffer system also gets derivatized. Simplifying the wash buffer helps with this, but you should be aware if there is any carryover derivatives in the wash buffer. The concept of a control is more related to comparing the metabolome between growth conditions. LC/MS is not quantitative because of ionization differences between individual molecules. Therefore, when you are comparing LC/MS results between two culture conditions, all you get is a ratiometric measurement of what went up and what went down. You don't get an absolute concentration. You can only get to an absolute concentration by spiking samples, preferably with stable isotope versions of the metabolites that you want to track. see:
Method Isotopic Vector Angle Analysis in Mass Spectrometry v2
- Badges
- Science method