High-sensitivity amino acid analysis by derivatization with o-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection: Applications in protein structure determination

Abstract

An amino acid analysis method using a commercially available analyzer that accurately quantitates protein-derived amino acids in the 10–100 pmol range is described. The method utilizes the robotic capability of the analyzer's autosampler to perform precolumn derivatization of both primary and secondary amino acids with o-phthalaldehyde and 9-fluorenylmethyl chloroformate, respectively. The derivatized amino acids are then separated on a C-18 reverse-phase amino acid column and quantitated in a single run by fluorescence detection. The characterization of β-lactoglobulin and two tryptic peptides from the bacterial enzyme diaminopimelic acid epimerase is used to demonstrate the sensitivity and utility of this method.