Chromatography Forum

Dear all,
We are using amino acid analysis (post column derivitization) for quantification of a certain purified protein to be used as an in-house reference, and I have a question about the calculations derived from such assay. As this assay uses a standard composed of quantified amino acids possible to detect and analyze by rhis method, the result is in terms of nmol (or pico mole). As we need to convert this to ug or mg, this is usually done by equations eg. 100 pmol of 100 kDa protein = 10 µg. In case of our protein, it a calculated molecular weight (based on it AA composition), but a bigger weight including its glycosilation. Which molecular weight should be used in such calculations? and can I find this explained somewhere on any website/publication?

Thank you

If I understood the question correctly, the answer would be "it depends" on whether you will be assaying the glycosylated or unglycosylated protein going forward. If you will be using the assay to determine the degree of glycosylation, for example, then the relevant molecular weight would be that of the unglycosylated protein.

If I understood the question correctly, the answer would be "it depends" on whether you will be assaying the glycosylated or unglycosylated protein going forward. If you will be using the assay to determine the degree of glycosylation, for example, then the relevant molecular weight would be that of the unglycosylated protein.

Dear Tom,
I am using the assay to quantify this in-house reference, and this in turn will be used as a reference for an HPLC-UV method for quantifying that protein in our finished product, would this clarify the aim of my question? To clarify, does amino acid analysis quantify only the amino acid part of the protein (which I think is the case) without the glycosilations/pegylations which anyway don't have absorbance in the UV region used in amino acid analysis? and if that's the case, would it be correct to use the total molecular weight (including the glycosilation) for converting the nmoles to ug derived from this amino acid assay?

that doesn't clarify things because I still don't know how your product is specified: as the "naked" protein or as a glycosylated protein. I suspect the latter, but if it's ambiguous, then you need to go back to whomever developed the product and find out for sure.

that doesn't clarify things because I still don't know how your product is specified: as the "naked" protein or as a glycosylated protein. I suspect the latter, but if it's ambiguous, then you need to go back to whomever developed the product and find out for sure.

If you search for our protein in databases, it is described by the molecular weight of the unglycosilated part (theoretical or calculated weight based on the amino acid composition), but in reality (for instance in papers where the protein is measured by ESI-MS), it is described with a bigger and approximate weight that includes the glycosilation.
Our final product of course includes the whole molecule (glycosilated), and we want to quantify the whole molecule in the product, my question is what does amino acid analysis AAA quantify? so if AAA has a result that my sample contains 100 pmol, is this 100 pmol of naked protein or the glcosilated protein?
Please excuse my questions, as I don't have hand on experience with AAA, and we are trying to careful with data generated with this method.

is this 100 pmol of naked protein or the glcosilated protein?

It's both. 100 picomoles is 100 picomoles. That is independent of the molecular weight. It's only when you want to convert moles to mass that molecular weight enters into it.

This comes up more frequently with basic drugs which are often available as the "hydrochloride salt". You could report the mass of the free base or the mass of the salt. Which one you use depends on how the specifications are written. So long as the spec is unambiguous ("100 micrograms of dammitol free base" or "100 micrograms of dammitol hydrochloride" per tablet) it really doesn't matter.