Chromatography Forum

Hi everyone

I am new to the field of Amino acid analysis. Can somebody suggest me the equipment to perform acid hydrolysis of peptides for amino acid analysis. Additionally if somebody can suggest some good reading material (including books) for the analysis of peptides.

Thanks

There are several posts in this forum concerning AA analysis. You may try a search for those.

Depending on the detector you have, you can choose the most adequate HPLC method for you.

Both proteins and peptides are normally hydrolyzed using vey pure 6N HCl in sealed vials from which all oxygen has been removed by vacuum.
Waters had a method called "Pico-Tag" which had the necessary equipment. CEM also has a microwave digestion system which is reported to work well. There is a lot of information available with a little searching. One good place is the Association of Biomolecular Resource Facilities or "ARBF". Do a web search. There was an amino acid tutorial posted there in the past. You can also contact Phenomenex for a copy of their User's Guide to "Introduction to peptide and protein HPLC". Good luck with your searches!

You might also look in J. AOAC Intl. They have published several methods with collaborative validations studies. Their methods are aimed at characterizing food products, but the principles are sound.

The method with 6N HCl is the "classic" of protein hydrolysis. In addition to the Water kit, Pierce sells a nice one. Other methods have been developed over the years to handle specific issues. The classic method has essentially no recovery for tryptophan, and some loss of serine. Oxidation of sulfur amino acids can be a problem.

Variations: Methanesulfonic acid instead of HCl; supposedly better recovery of sulfur amino acids. Alkaline hydrolysis; recovers tryptophan. Pre-oxidation with H2O2/CH3CCOOOH/etc; quantitative conversion of methionine to the sulfone and cysteine to cysteic acid. HCl + phenol; for folks without fancy vacuum hydrolysis equipment.

Once upon a time, when amino acid analysis was really high-tech, there were vapor-phase hydrolysis devices for small protein/peptide samples.

I think that Fountoulakis has published a paper where he compares different protein digestion protocols.

I have the paper, but I will need to find it...

On the Waters website you should find several methods for AAA. Besides the Pico-Tag method, you will find the AccQ-Tag method, which may have some advantages for you.

Another great resource for AAA is at www.abrf.org They emphasize research and pharma applications.

Thank you all for the great suggestions. I am getting acquainted with the subject.

Pawan,

Here is the review article I menioned in my previous post: Fountoulakis and Lahm, Hydrolysisi of amino acid composition analysis of proteins, J. Chromator. A 1998, 826, 109-134.

Good stuff!