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 and Methionine are not damaged during the hydrolysis procedure. In order to preserve any Tryptophan, samples are put through an alkaline hydrolysis. 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 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 also prepared using an alkaline hydrolysis method to generate Tryptophan. Samples are then diluted to an appropriate concentration based on the protein content of the sample. For the samples having undergone acid-stable hydrolysis and performic acid pretreatment hydrolysis, 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. For samples having undergone alkaline hydrolysis, a solution of phosphate buffer is added to the sample to achieve the target dilution. All sample mixtures are then filtered into an HPLC vial.

Hydrolyzed amino acid analysis (acid-stable and performic acid pretreatment hydrolyzed samples only) 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.

Alkaline hydrolyzed tryptophan amino acid analysis is performed using a reversed phase C-18 column with an aqueous buffer containing organic solvents using an HPLC analyzer.  Peak detection uses a combination of in-line, programmable, and scanning fluorescence detectors. 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 utilizing a calibration curve.

Amino Acids Quantified

Hydroxyproline

Glutamic Acid

Phenylalanine

Cysteine/Cystine

Aspartic Acid

Proline

Isoleucine

Lysine

Methionine

Threonine

Glycine

Leucine

Histidine

Tryptophan

Serine

Alanine

Valine

Tyrosine

Arginine