I need to check whether the level of metabolites are altered in cell culture media during specific treatment condition because of altered uptake and secretions.
Don't be in a hurry!
First thing is to become familiar in general with your LC/MS system. Next become familiar with standards based on the analytes that you'll be investigating; appropriate HPLC conditions, and optimum settings on the mass spectrometer. Make sure you can reproduce retention times and MS responses. Spike your media with standards to determine extraction efficiencies. Find good internal standards. Get a sense of the precision of your system, e.g., how much variation do you see if you inject the same sample several times in a row; then how much variation from morning to evening; then how much variation between Monday and Wednesday; etc.
In order to determine if there are differences in metabolite levels for different experimental conditions, you need to be confident that you can: 1. detect which of the many metabolites have changed and 2. quantitate the changes.
What you're planning isn't particularly easy, but it's certainly do-able. Just make sure that you do your homework before doing the actual experiments.
Best of luck in this!
Regards.
Pete
If you have an HPLC or GC method for the metabolites, I'd use those to resolve the metabolites with the MS as a detector, along with the detector on the chromatography system (UV, FID) for the compounds.
It depends somewhat on the nature of the metabolites - are there a particular type of metabolite you are looking for?
dear dr. silver,
thank you for your reply. we have hplc coupled with MS/MS system. but no chromatography detectors. its a new setup and we havent used it yet.
Dear Dr. Kaplan,
i am looking for the change in uptake of the metabolites and media components from cell culture media. basically carbohydrates.
Don't be in a hurry!
First thing is to become familiar in general with your LC/MS system. Next become familiar with standards based on the analytes that you'll be investigating; appropriate HPLC conditions, and optimum settings on the mass spectrometer. Make sure you can reproduce retention times and MS responses. Spike your media with standards to determine extraction efficiencies. Find good internal standards. Get a sense of the precision of your system, e.g., how much variation do you see if you inject the same sample several times in a row; then how much variation from morning to evening; then how much variation between Monday and Wednesday; etc.
In order to determine if there are differences in metabolite levels for different experimental conditions, you need to be confident that you can: 1. detect which of the many metabolites have changed and 2. quantitate the changes.
What you're planning isn't particularly easy, but it's certainly do-able. Just make sure that you do your homework before doing the actual experiments.
Best of luck in this!
Regards.
Pete
Dear Dr. Wishnok,
thank you very much for your suggestion. i sure was in a hurry but now i understand its not as easy as i thought it will be.
you are an expert on the mass spectrometry based metabolite analysis and metabolite profiling and i would like to keep writing to you regarding the theoretical and technical problems i will face.
thank you again for your enlightening reply.
regards
milton
are you planning on extracting? if so what method will you be using? What type of MS do you have available?
Dear Dr. Kaplan,
we have ABI Sciex 3200 QTRAP system. I was thinking about simply running the culture media through LC/MS, but the idea of extraction sounds interesting. although extraction of carbohydrates from aqueous base media is not easy but there are reports suggesting that it is possible. i just have started working on it. hope this or some other method will work out. and not 'just carbohydrates but TCA cycle intermediates are my interest, so if you have any suggestions please write.
regards
milton
Although some do it, it's not the best idea to inject raw culture media into the LC/MS system. The cleaner your samples, the longer the MS will stay clean with maximum sensitivity. Check the literature on the analysis of carbohydrates and TCA-cycle metabolites. I have limited experience with this, but do know that these analyses are non-trivial and typically require non-routine LC methods, e.g., HILIC columns (which are tricky). You may want to consider 2D methods for clean-up.
Here are some free online references (there are many more out there, depending on subscriptions available to your institution):
http://www.chromatographyonline.com/lcgc/article/articleDetail.jsp?id=429510
http://link.springer.com/article/10.1007/s00216-008-2060-6#page-1
http://link.springer.com/article/10.1007/s00216-011-5308-5#page-1
http://www.chromatographyonline.com/lcgc/article/articleDetail.jsp?id=667489
http://www.dionex.com/en-us/webdocs/110521-PO-IC-Carbohydrates-21Nov2011-LPN2954-01.pdf
Dear Dr. Wishnok,
thank you for the links. really useful it is. I have got some literature on analysis of TCA cycle intermediates from tissue sample using methanol water extraction, but for carbohydrates HILIC columns sounds interesting. I will look more into literature and then will plan my work. i will ask for suggestions if i find some difficulties in LC/MS/MS method.
regards
milton
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