PROFILING PEPTIDE ADDUCTS OF OXIDIZED N-ACETYLDOPAMINE BY ELECTROSPRAY MASS-SPECTROMETRY

Phenolic and catecholamine polymers are common constituents of many bi ological systems. Cross-linking of polyphenols with other phenols, pep tides, proteins and carbohydrates results in the synthesis of complex natural products which are not easily characterized. Electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (ES-MS/MS) were use d to identify polymers of oxidized N-acetyldopamine (NADA) and peptide adducts with oxidized NADA. Following incubation with mushroom tyrosi nase, NADA adducts of trityrosine were identified. It was not possible to locate the site of NADA binding to this tripeptide. Compounds form ed by incubation of N-acetylhistidine and N-acetyllysine with oxidized NADA, previously characterized using classical chemical techniques, w ere confirmed using ES-MS/MS. The peptide angiotensin (DRVYIHPFHL) was used as a model substrate to determine whether the site(s) to which o xidized NADA bound could be determined. The lot of angiotensin used wa s contaminated with a peptide of mass 14 u greater than angiotensin, a nd it was found that the H in position 9 of the contaminant peptide wa s modified. ES-MS/MS of the angiotensin and the contaminant peptide fo llowing incubation with oxidized NADA revealed that the C-terminal asp artic acid was the primary amino acid to which NADA adducts were coval ently bound, but other residues were also modified. Femto-molar sensit ivity for analysis of complex mixtures of catecholamine-peptide adduct s will facilitate structural elucidation of natural products not amena ble to characterization using other spectroscopic techniques. (C) 1997 by John Wiley & Sons, Ltd.