Chromatography Forum

Dear all,

New tasks, new challenges...

I want to quantify a peptide (nine AA's) which is a solution containing about 10 ng/ml with HPLC (not MS). This might be common knowledge, but is there a good fluorescent derivatisation reagent that could work here? I guess the best point of reaction would be in the carboxylic end of the peptide (?)

many thanks!

Oops, I didn't have all information when I wrote the question.

The peptide is already modified in the carboxylic end, so I guess that option is out of the question. Maybe the suphur bridge can be used instead?

Hi Mathias,

For good reasons, I don’t know your peptide’s amino acid composition, but if it contains one or more aromatic amino acids e.g. tryptophane, tyrosine, phenylalanine etc. you might as well measure the peptides intrinsic fluorescence. It makes the task so much easier

Best Regards

The peptide contains one phenylalanine.

Could you explain what intrinsic fluorescence is, and how to measure it?

many thanks
/Mattias

You can look at this article:

Anal Chem. 2004 Feb 1;76(3):728-35

Fluorogenic derivatization reagents suitable for isolation and identification of cysteine-containing proteins utilizing high-performance liquid chromatography-tandem mass spectrometry.
Masuda M, Toriumi C, Santa T, Imai K.

Hi Mathias,

Intrinsic fluorescence is just the ability of a molecule to fluoresce by its self. So if your peptide contains aromatic amino acids, which all fluoresce, then you can utilize this property and measure the light emitted upon excitation of the molecule.
You can typically excite aromatic amino acid containing proteins/peptides at 280 nm (all depending on pH and temperature) and measure the emission at about 320 – 370 nm.
But remember to determine the excitation and the emission wavelengths more accurately, bearing in mind your need for all sensitivity you can get, due to the small amount of material you have, as I understand.

Best Regards

Thank you both!

I have ordered the article now, and I will let you know how it goes!

I will try to use the intrinsic fluorescence properties first, but I doubt that the quantum yield is good enough. That would of course be the easiest solution!

Yes, I would also be surprised if the quantum yield was high enough. There are lots of proteins, though, that fluoresce fairly well. They have their rigid aromatics held relatively rigidly in a low polarity area of the protein.

How about FMOC-CL on an amino end?

If your peptide has a free amino end, you could use the AccQ-Tag reagent from Waters. It reacts with primary and secondary amine functions with a very rapid (< 1 min) reaction time to give a stable fluorescent product.

Uwe> Sounds promising! A downloaded the application from Waters homepage, but there was no explaination to the the term "pre-column derivatisation". How does that work?

Pre-column derivatization primarily means that it is not a post-column derivatization method, as the classical amino acid methods were. You can carry this pre-column derivatization reaction either out automatically, i.e. the injector does the job for you by mixing the analyte and the reagents, or simply in the vial, and you mix the stuff yourself.

OK, it was less complicated than I thought... It is a difference between precolumn and pre-column!

I thought they meant that the reaction could take place while the analyte was adsorbed on a trap column.

Do you have any numbers of the absorption/fluorescence of the Acc-Q tag? I looked up some reagent called Alexa-fluor that has a very strong absorption/fluorescence. But yours seems easier to work with at first glance.

The information in the catalogue says that you can comfortable do an amino acid analysis with 0.02 pmoles in 10 microl. Assuming that you peptide has a molecular wweight of about 1000, this gets you readily into the range that you desire, even for a 10 microL injection.

Uwe> I have now done the first test with the AccQ kit, and the chromatogram looks quite noisy. I see the AMQ peak early in the chromatogram, but then there are at least three major peaks eluting after.

Will the AccQ react with other sites than the N-terminus? I suspect that I see multiply labelled molecules.

It reacts with primary and secondary amines. Do you have more than the N-terminal with such functions?