I want to check the change of metabolites in Cyanobacteria after supplementing glucose in the media. I am interested to see the metabolites of Glycolytic, TCA cycle and OPP pathways. Which methods would be suitable for that?
You want 1) a chromatographic system capable of separating your metabolites. RP-HPLC is the usual choice for polar metabolites, but I have had a lot of success separating TCA cycle intermediates by capillary zone electrophoresis. (In my opinion, CZE is an excellent technique which isn't used nearly often enough). 2) A software package capable of multivariate statistical analysis (i.e. ANOVA and Principal Component Analysis), because you will be measuring changes in peak areas and you want to determine which changes are statistically significant. R is a good package, and it is free, but you have to learn the programming language. I believe that the makers of Sigma Plot have statistical software available which can perform ANOVA and PCA, but you will have to pay it out of your funding if your institution doesn't already have it.
As you are interested in three particular metabolic pathways, you might consider feeding your cyanobacteria with isotopically labelled substrates. Then the target metabolites will be easier to detect. If you grow your cells in media containing labelled glucose, for example, you will be able to follow the label through the glycolytic and TCA cycles. Whether to use radioactive or stable isotopes is another question. Radioactive compounds increase the detection limit, but require dedicated lab space, equipment, and special procedures for disposal. Substrates labelled with stable isotopes may be more expensive, but they can be detected easily on HPLC-MS. (There is software for that too: FiatFlux is supposed to be very good).
P.S. I could also have mentioned GC-MS, as a chro;atographic technique, but it is more typically used for non-polar compounds such as oils and steroids. Polar compounds need a derivatisation step to convert them to hydrophobic analogues.
@Sally Burr , Thanks for your very informative suggestion. It seems you are very skilled in this area. Would you mind if I request you to give me the protocols you usually follow to prepare the sample for RP-HPLC and CZE?
Thank you again
Rezaul
I am less familiar with sample preps in bacteria than in plants, seeing as I have done all my metabolism work in Arabidopsis, Medicago truncatula and maize. My protocols generally involved a rapid freezing step using liquid nitrogen, followed by homogenisation (ball bearings in eppendorf tubes, or the pestle and mortar for large samples) followed by extraction of solubles (i.e. with 70% methanol), filtration, then HPLC.
Alternatively all proteins and cell wall polysaccharides would be precipitated with ethanol, and the supernatant was washed with ethyl acetate to remove non-polar compounds (fatty acids, etc) and then passed through ion exchange resins to separate neutral polar compounds (including sugars) from positively charged amino acids and negatively charged organic acids (TCA cycle intermediates).
The filtration step is vital for HPLC analysis, because particles can cause blockages in the apparatus. A length of CZE column can be replaced quickly, but the HPLC system may have to be taken apart completely.
(Although it's probably no use to you I can't resist mentioning an extraction technique I learnt from Professor Deri Tomos in Bangor for extracting all vacuolar liquid from a single seedling. Simply place the seedling in an small eppendorf and dip in liquid nitrogen, then quickly pierce the end with a needle, put it in a larger eppendorf and centrifuge. You will get 10 or 20µl of aqueous fluid, which is ready for analysis right away.)
You, on the other hand, probably want a quick bacterial lysis step, followed by precipitation and centrifugation. You may want to pellet your bacteria first in the centrifuge, then resuspend them in a small volume of water. That would avoid any problems due to salts and buffers (which would interfere with the chromatgraphy of the organic acids). If you include washing steps (repeated resuspensions in water to get rid of unused labelled substrate) you might want to check your sample under the microscope to see if there is any lysis going on before you want it to happen.
After that, if your sample is large enough, you might try clean up with ion exchange resins as above (which may be available as cartridges for quantities of 1ml or under). You have to bear in mind that you will lose a small amount of sample at every preparation step, so when you are devising your own protocols you need to find the right balance between sample purity and having enough analyte to detect.
Hope this is helpful.
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