Detection of peptidyl-3,4-dihydroxyphenylalanine by amino acid analysis and microsequencing techniques

3,4-Dihydroxyphenylalanine (DOPA) is an amino acid that occurs naturally in the primary sequence of many proteins and peptides. Detection of peptidyl-DOPA, however, can be elusive. This is due (i) to its coelution with leucine on most of the ion exchangers used in amino acid analysis and (ii) to the coelution of phenylthiohydantoin (PTH)-DOPA with PTH-alanine during routine C18 reversed-phase HPLC following automated Edman degradation. By application of appropriately timed temperature and/or gradient modifications during chromatography, DOPA and its PTH derivative can be adequately resolved for detection by both amino acid analysis and gas-phase sequencing.