Endogenous 3,4-Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine: LINKS TO MITOCHONDRIALLY DERIVED OXIDATIVE STRESS VIA HYDROXYL RADICAL*

Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3,4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first proteome survey of endogenous site-specific modifications, i.e. DOPA and its further oxidation product dopaquinone in mouse brain and heart tissues. Results from LC-MS/MS analyses included 50 and 14 DOPA-modified tyrosine sites identified from brain and heart, respectively, whereas only a few nitrotyrosine-containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 20 and 12 dopaquinone-modified peptides were observed from brain and heart, respectively; nearly one-fourth of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal binding properties, consistent with metal-catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondrially associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation, suggesting potential disruption of signaling pathways. Collectively, the results suggest that these modifications are linked with mitochondrially derived oxidative stress and may serve as sensitive markers for disease pathologies.Generation of reactive oxygen species (ROS)¹ and reactive nitrogen species is a normal consequence of aerobic metabolism that, in excess, results in oxidative stress that further leads to oxidative modification of proteins, lipids, and DNA, events that may lead to altered cellular function and even cell death (1, 2). Chronic oxidative stress is well recognized as having a central role in disease and is responsible for both direct alteration of biomolecular structure-function and compensatory changes in cellular processes (1–4). It is increasingly recognized that oxidative modifications of proteins can serve as potential biomarkers indicative of the physiological states and changes that occur during disease progression. Thus, the ability to quantitatively measure specific protein oxidation products has the potential to provide the means to monitor the physiological state of a tissue or organism, in particular any progression toward pathology. Given Parkinson disease (PD) as an example, a number of oxidative modifications on proteins pertinent to PD have been identified, further supporting the potential importance of oxidative modifications to disease pathogenesis (5).Many oxidative modifications on specific amino acid residues, such as protein carbonylation (6), cysteine S-nitrosylation (7–9), cysteine oxidation to sulfinic or sulfonic acid (10–12), methionine oxidation (13, 14), and tyrosine nitration (15–21) within complex protein mixtures, have been detected by MS-based proteomics; however, their low abundance levels within complex proteomes often hinder confident identification of these potentially significant modifications (22). For example, tyrosine nitration is a well studied post-translational modification mediated by peroxynitrite (ONOO⁻) or nitrogen dioxide (^·NO₂), which commonly occur in cells during oxidative stress and inflammation; however, only a small number of nitrotyrosine proteins have been identified from a given proteome sample because of insufficient analytical sensitivity and the chance of incorrect peptide assignments (19, 23). With recent advances in high resolution MS that provide high mass measurement accuracy, the ability to confidently identify modified peptides has been significantly enhanced (24).Hydroxyl radical (HO^·) is one of the most reactive and major species generated under aerobic conditions in biological systems (1, 25, 26). Among several HO^·-mediated oxidative modifications, the protein tyrosine modification 3,4-dihydroxyphenylalanine (DOPA) has been reported as a major product and index of HO^· attack on tyrosine residues in proteins (Fig. 1) (27, 28). DOPA is also formed on protein tyrosine residues via controlled enzymatic pathways through enzymes such as tyrosinase or tyrosine hydroxylase (28). Once formed, protein-bound DOPA has the potential to initiate further oxidative reactions through binding and reducing transition metals or through redox cycling between catechol and quinone (dopaquinone) forms (29, 30). Recent studies have suggested that protein-bound DOPA is involved in triggering antioxidant defenses (30) and mediating oxidative damage to DNA (31). Moreover, elevated levels of protein-bound DOPA have been reported in several diseases, including atherosclerosis, cataracts, and myocardial disease, and in PD patients undergoing levodopa therapy (26, 32–36). However, the specific DOPA-modified proteins, which could provide mechanistic knowledge of the progression of these diseases, have not been identified (27, 28). The ability to identify site-specific protein modifications should lead to a better understanding of the role of DOPA modification in disease pathologies as well as new molecular signatures or therapeutic targets for diseases.Open in a separate windowFig. 1.DOPA and dopaquinone formation from tyrosine.Therefore, in this study, we demonstrate the ability to identify site-specific DOPA and dopaquinone (DQ) modifications on protein tyrosine residues in normal mouse brain and heart tissues and their relative stoichiometries that are present in vivo under non-stressed conditions. Such endogenous protein modifications were detected using LC-MS/MS. The results from this global proteomics survey suggests that HO^· in tissues under normal conditions is generated largely from the mitochondria and metal-binding proteins where the resulting DOPA/DQ modifications have the potential to disrupt mitochondrial respiration as well as alter tyrosine phosphorylation signaling pathways such as 14-3-3-mediated signaling in brain tissue.