Development of fluorescent/stable isotope tagging strategies for proteins containing DOPA

Oxidative modification of protein tyrosine, including hydroxylation, to form proteins containing DOPA (PC-DOPA), has been associated with many age dependent pathologies. To date, there has been no report of a chemically selective method for in-vivo determination of PC-DOPA. By building upon previous derivatization methods that used benzylamine (BA) or diphenylethylenediamine (DPE) to form highly fluorescent products with catechols and catecholamines, a fluorescent/stable isotope tagging strategy for PC-DOPA was developed. By examination of the products derived from the reaction of small molecule model compounds with DPE and BA reagents, a robust understanding of the reaction process leading to the formation of highly fluorescent substituted benzoxazole products was established. These products were identified by a combination of MS, multi-dimensional NMR, and X-ray crystallography. With this information in hand, it was determined that the use of BA would be more efficient for experiments where isotopic labeling was required, i.e., use of BA with 5 deuterium atoms substituted on the aromatic ring for relative quantitation by MS. Relative quantitation was demonstrated using 4-methylcatechol and DOPA-G-G as model compounds.