Analysis of Polypeptides by Amino Acid Analysis.

ABSTRACT

Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.