I would like to characterise how much of various (uncommon) cytosolic amino acids are produced in bacteria, and was wondering if there are good suggestions of how to go about doing this. I know that chromatography is a good end-point analysis once I have isolated a fraction of what I suspect contains the amino acids; however, I am unsure about the smartest way to do the 'rough' initial purification prior to chromatography.

I know that you in general lyse the cells, pellet cell debris and then start some form of purification. I have also heard of certain resins that can bind natural products (e.g. Diaion® HP20, synthetic adsorbent resin) had thus aid the first purification step of the amino acids. Do people have experience with this? Are there also good solvents for separation of amino acids from lysate, that could be used in the first step (phase separation)? Does someone have any experiences with this? Any alternative suggestions would be appreciated.

  • $\begingroup$ Please clarify what you are trying to characterize. 1) You seem to be using amino acid and "natural product" as if they were synonymous, but amino acids are only one type of metabolite that you could call a "natural product". IMO "natural product" isn't a helpful categorization since it could refer to anything made by an organism — do you have a more specific definition? ——— 2) Do you have a defined list of what you want to characterize or are you interested in "fishing" for all molecules that fit certain criteria? $\endgroup$ – tyersome Jun 20 at 19:49
  • $\begingroup$ Yes, amino acids of various kinds (common and non common). I thought this would be specific enough? Amino acids is a type of molecule with the amine and carboxylate group on each end, and various side chain (depending of which amino acid it is). Overall they are very small molecules (under 300 g/mol). $\endgroup$ – CuriousTree Jun 22 at 8:49
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    $\begingroup$ I edited your post to something I find clearer — please edit if you feel that I've changed the essence of your question. Is there some reason you picked Diaion HP20? I had a quick look at the Sigma page for that resin and its description doesn't sound promising to me ... $\endgroup$ – tyersome Jun 23 at 1:04
  • $\begingroup$ Diaion HP20 is simply the only one I have head of, and the reasons I was asking what people think about it (or perhaps have head of something better?). Thanks for the edit - this is much better! $\endgroup$ – CuriousTree Jun 23 at 6:39
  • $\begingroup$ It sounds like you'll need to do a protein hydrolysis reaction to seperate the cellular proteins into individual amino acids. This book chapter compares different methods and their various pros and cons (link.springer.com/protocol/10.1385/1-59259-342-9:111). If you don't have institutional access, just find a paper that does a similar analysis to the one you want, and reference the methods. Just make sure they did a decent job of validating it before you used it. Published method does not always equal valid method. $\endgroup$ – MikeyC Jun 23 at 15:15

A good approach could be to use GC-MS to identify cytosolic metabolites. When focussing on cytosolic amino acids, it might be useful to precipitate the proteins in a purification step and continue with the soluble fraction. Subsequently, a derivatization is done. The mass spectrometry helps to better identify the amino acids. This article by Phelippe et al. can give a good start.

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  • $\begingroup$ Thanks, this was exactly the type of literature I was looking for! I filtered the lysate solution and I am now attempting a freeze-dry, to then resuspend it in some type of solvent that proteins dont like (might be a good way to completely remove them) - before e.g. GC-MS. I will have a look at the paper! $\endgroup$ – CuriousTree Jul 1 at 6:37

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