Amino Acid Analysis
An analytical technique that determines the amino acid composition of a peptide by hydrolysing the peptide bonds and quantifying each amino acid present. AAA confirms that a synthesised peptide has the correct overall composition, complementing sequence-specific methods like mass spectrometry.
Technical Context
AAA procedure: (1) acid hydrolysis (6N HCl, 110°C, 18-24 hours — breaks all peptide bonds, but destroys Trp completely and partially degrades Cys, Met, Ser, Thr), (2) derivatisation (converting free amino acids to UV/fluorescent derivatives for detection — pre-column: OPA, FMOC, AQC, dansyl chloride; post-column: ninhydrin), (3) separation (RP-HPLC or ion-exchange chromatography), and (4) quantification (comparing peak areas to amino acid standards). Results: molar ratios of each amino acid compared to theoretical composition. AAA confirms overall composition but NOT sequence (Gly-Ala-Ser gives identical AAA results to Ser-Gly-Ala). For Trp quantification: alkaline hydrolysis (4M methanesulfonic acid, which preserves Trp) or spectrophotometric estimation is used. For Cys: performic acid oxidation (converting Cys to cysteic acid, which is stable to acid hydrolysis) is used before hydrolysis. AAA is also used for peptide content determination — quantifying the actual peptide mass per vial.