I have liver and serum results from metabolomics analysis. Some of the differential signals were identified as amino acids. I am curious whether the amino acids originated from actual metabolism pathways or just fragments of proteins that are not completely removed.
Lin,
The origin of the amino acids can not be determined by LC-MS/MS, but the presence of amino acids that originates from proteins could be limited in your sample preparation with the use of protease suppressors.
Look for upstream metabolites of the corresponding amino acids, for instance serine increase should also give you an increase of O-phosphoserine, 3-phosphohydroxypyruvic acid ... . It is not waterproof, but at least some extra evidence for your data interpretation.
I guess this is not applicable for your research, but if you are working with cultured cells you might think of adding 13C glucose or other 13C tracers to your cells, if protein synthesis occurs, some of the amino acids should contain incorporated 13C atoms (not for all amino acids!). You will need to look into the pathways of those specific amino acids to figure things out.
If you think your extraction method is too harsh, you could try filtration instead.
In principle, your proteins are denaturated during extraction, including proteases.
I agree with ma Stander, in LCMSMS it's not possible to distinguish the origin of the aminoacids. If you want to measure only intracellular or extracellular aminoacids and not those incorporated in proteins, you have to ensure the purity of your sample prior to LCMSMS analysis. I think thar using 13C tracers can be a solution only if protein synthesis doens't occur. When using 13C tracers, the tracer is incorporated in the metabolites, including aminoacids. If protein synthesis occurs, recruiting these aminoacids, the aminoacids in the protein will also be traced with 13C...
Ma Stander said it right. If your proteases are inhibited, the amino acids can not come from proteins. However, do not assume that your extraction protocol will denature all proteins if you have not boiled your samples or added protease inhibitors. We typically use a cocktail of inhibitors to inhibit all types of proteases: aprotinin, pepstatin, chymostatin, EDTA, PMSF, antipain and leupaptin.
Metabolomics will give global picture of metabolites only. To know the origin, you have to go through hypothesis derived biochemistry. Experimental design will depend on your hypothesis.
The LC-MS/MS can not determine the origin of amino acids. Also proteases in use do not inhibit carboxy and aminopeptidases. You may try to remove any protein or peptide contamination using high TCA concentration for precipitatation or by using filtration
Well not metabolomics will not tell you the origin of you aminoacids but you can surely avoid proteolisis and then and easy step to d is to precipitate all your proteins present in your samples including proteases that could be done using MeOH 80 or 90 % all proteins will precipitate an you can separate them by centrifugacion, small molecules such as aminoacids will remain solubles.
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