Purpose

Samples containing peptides and proteins can be hydrolyzed using an acid stable hydrolysis procedure to release the bound amino acids. This method is for accurate determination and quantification of amino acids in proteins.

Summary

Samples are digested using an acid-stable hydrolysis procedure to release the bound amino acids present in the protein of the sample. Samples are then diluted to an appropriate concentration based on the protein content of the sample. A pre-made mixture of a deproteinizing solution (5-sulfosalicylic acid hydrate), internal standard (L-2-Amino-3-Guanidinopropionic Acid Hydrochloride), and sodium buffer is added to the sample to achieve the target dilution. The mixture is then filtered into an HPLC vial.

Hydrolyzed amino acid analysis is performed on amino acid analyzers. Amino acids are separated by cation exchange chromatography using various sodium eluents. Absorbance is measured at 440 and 570 nm following post-column color development by Ninhydrin reagent. Appropriate standards are run to ensure each sequence is running properly and are used in the quantification of samples using peak areas and applying an external standard technique.

Limits of detection are 0.3 to 1.0 micromoles per liter. There is no high-end limit as the sample can always be diluted down to an appropriate detection range.

Amino Acids Quantified

Hydroxyproline

Glutamic Acid

Phenylalanine

Aspartic Acid

Proline

Isoleucine

Lysine

Threonine

Glycine

Leucine

Histidine

Serine

Alanine

Valine

Tyrosine

Arginine