Profiling peptide adducts of oxidized N-acetyldopamine by electrospray mass spectrometry.

Abstract

Phenolic and catecholamine polymers are common constituents of many biological systems. Cross-linking of polyphenols with other phenols, peptides, 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 used to identify polymers of oxidized N-acetyldopamine (NADA) and peptide adducts with oxidized NADA. Following incubation with mushroom tyrosinase, NADA adducts of trityrosine were identified. It was not possible to locate the site of NADA binding to this tripeptide. Compounds formed by incubation of N-acetylhistidine and N-acetyllysine with oxidized NADA, previously characterized using classical chemical techniques, were confirmed using ES-MS/MS. The peptide angiotensin (DRVYIHPFHL) was used as a model substrate to determine whether the site(s) to which oxidized NADA bound could be determined. The lot of angiotensin used was contaminated with a peptide of mass 14 u greater than angiotensin, and it was found that the H in position 9 of the contaminant peptide was modified. ES-MS/MS of the angiotensin and the contaminant peptide following incubation with oxidized NADA revealed that the C-terminal aspartic acid was the primary amino acid to which NADA adducts were covalently bound, but other residues were also modified. Femto-molar sensitivity for analysis of complex mixtures of catecholamine-peptide adducts will facilitate structural elucidation of natural products not amenable to characterization using other spectroscopic techniques.