Amino acid analysis
Amino acid analysis determines the amino acid composition of a peptide or protein. It is also one of the most commonly used technique for quantitating proteins and peptides that does not depend on specific structural features of the polypeptide, like the conventional dye-binding methods. Amino acid analysis is often employed to asses coupling of peptides to carrier proteins like KLH; the quantitation is aided by incorporating a non-naturally occurring amino acid, such as norleucine or ß-alanine, in the peptide.
To start amino acid analysis, the polypeptide is normally hydrolysed in 6N hydrochloric acid vapor for 24 hours at 110°C. The free amino acids are then derivatized either with phenylisothiocyanate to produce phenylthiocarbamyl (PTC) amino acids. The derivatized amino acids are separated on a C18 reversed phase column with an acetonitrile gradient in sodium acetate buffer.
Sample Requirements
We need 2 to 10 µg of purified protein or peptide sample, preferably in a volatile solvent or solid. However small amounts of non-volatile salts can be tolerated. We encourage potential users to include a buffer solution for control.
Limitations
During acid hydrolysis, asparagine and glutamine are deamidated to aspartate and glutamate, respectively, rendering the acid and its corresponding amide indistinguishable. Tryptophan is destroyed under the standard hydrolysis conditions, and so is cysteine although to a lesser extent. Therefore special hydrolysis conditions are required for these two amino acids and they need to be carried out separately.