Purpose

Samples containing peptides and proteins can be hydrolyzed to release the bound amino acids. By doing a performic acid pretreatment on the protein, this ensures that the sulfur containing amino acids Cysteine/Cystine and Methionine are not broken during the hydrolysis procedure. This method is for accurate determination and quantification of Cysteine/Cystine and Methionine 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 also put through a performic acid pretreatment to add a sulfone protecting group on Cysteine/Cystine and Methionine present in the sample, generating Cysteic Acid and Methionine Sulfone before applying an acid hydrolysis procedure. Samples are then diluted to an appropriate concentration based on the protein content of the sample. A solution 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 HPLC 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 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

Cysteine/Cystine

Methionine