Assuming you want to quantify soluble protein, you can proceed with your preferred method for preparing crude lysate for protein purification (which will include a mild detergent to remove lipids, Benzonase treatment followed by GF to remove nucleic acids and other small molecule contaminants, etc.). Following, you need to acid hydrolyze your protein prep and derivatize the amino acid. Make sure to include a control of known protein concentration while optimizing your method to make sure you are not loosing protein.

As a last note, this is quite a challenging project for a first time HPLC user, I recommend you find expert help locally if possible. If not, come back here.

Thank you Carlo for your answer!

I think I didn't explain properly what I plan to do. I will supply E. coli with an unnatural amino acid in the growth medium and I only need to check if the bacteria take it up. So I roughly want to quantify soluble amino acids inside the cells. I know about one unnatural amino acid that they take up sufficiently well enough which I would determine as a positive control. I hope it is understandable now what to I mean.

I expect that after preparing cell lysate I have to remove proteins, nucleic acids, lipids and so on in order to get pure cytoplasm but I don't know how to perform this step.

The difficulty here is extract only amino acids from the crude extract which is very difficult. A quick way to have precipitate most of the non metabolite there is simply add acetonitrile to the crude extract, vortexing, then spin at 5000rpm and use the liquid (you could repeat the extraction from the solid). However this liquid contain a good deal of non aa metabolites which is soluble and will interfere at some point.

Thank you for your answer! I will have to try around a little bit I guess. How much acetonitrile is added roughly? About 1:1 to the buffer?