Development of analytical tools to enrich and identify protein dopaminylation

Abstract

Dopamine homeostasis and oxidative stress are thought to be key players in the etiology of Parkinson’s disease (PD). PD is a disorder characterized by the death of dopaminergic neurons in the substantia nigra and the formation of protein aggregates containing α-synuclein. Aberrant cytosolic dopamine levels have been linked to mitochondrial and lysosomal dysfunction, oxidative stress, and increased expression of α-synuclein in dopaminergic neurons. Outside of vesicles, dopamine is rapidly oxidized and accumulates in cells as various polymerized forms including neuromelanin. Oxidized dopamine can also covalently modify nearby proteins leading to structural and functional consequences, including aggregation. Currently, little is known about dopaminylation of proteins as dopamine is not widely recognized as a post translational modification. While several studies have identified dopamine-protein adducts, these studies are limited in scope or rely on a priori knowledge of likely candidate proteins due to lack of relevant discovery tools. Here, we describe several non-targeted approaches to investigate dopaminylation, including a method for enrichment, identification, and site-localization of dopaminylated peptides. Our method uses m-aminophenylboronic acid resin for covalent capture of the cis-diol moiety of dopamine to enrich dopaminylated peptides followed by identification using tandem mass spectrometry. Using this strategy, we identify over 1800 dopaminylated peptides from cultured cells and 20 dopaminylated peptides from post-mortem human brain tissue. In both cultured cells and brain tissue, we identified dopaminylation at Cys147 in SOD1. SOD1 is a key protein in oxidative stress and neurodegeneration, and modifications of Cys147 promote protein dysfunction and aggregation. We believe these approaches to be powerful tools to characterize site-specific dopaminylation of proteins, enabling mechanistic studies into the role of dopaminylation in the pathogenesis of Parkinson’s disease.