Complete automatization of peptide maps by reversed-phase liquid chromatography using o-phthalaldehyde pre-column derivatization☆

Abstract The conventional automatic o- phthalaldehyde (OPA) pre-column labelling procedure used for routine amino acid analyses by high-performance liquid chromatography has been applied as a highly sensitive technique for peptide mapping. Tryptic peptide digests are first derivatized with OPA by using an automated pre-column derivatization system and then the fluorescent peptide mixture is automatically analyzed by reversed-phase HPLC. The complete automatization of peptide mapping is achieved in about 45 min with a high resolution and a routine sensitivity in the range of 10–30 picomoles. Peptide map analyses of dansylated tryptic peptides using the above automatic conditions and with an equivalent range of sensitivity are also described. The effectiveness of this method has been exemplified by using two proteins which are nearly identical in their amino acid sequences.