Endogenous 3,4-dihydroxyphenylalanine and dopaquinone modifications on protein tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical

Abstract

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.

Fig. 1.

DOPA and dopaquinone formation from tyrosine.

Fig. 2.

MS/MS spectra of non-modified (top), DOPA- (middle), and DQ-modified (bottom) peptide VVAGVATALAHKYH from hemoglobin subunit β (Hbb-b2) in mouse heart. Y*, DOPA-modified Tyr; Y#, DQ-modified Tyr.

Fig. 3.

Spectral counts of DOPA- and DQ-modified proteins identified from mouse heart tissue before and after chemical treatment by iron/EDTA/ascorbate system. Dopaquinone_Ctrl, DQ-modified proteins from control sample; Dopaquinone_treated, DQ-modified proteins from treated sample; DOPA_Ctrl, DOPA-modified proteins from control sample; DOPA_treated, DOPA-modified proteins from treated sample.

Fig. 4.

Preferential modifications on mitochondrial and cytoskeletal proteins from brain (A) and heart (B). Gene ontology analysis of the cellular locale of DOPA/DQ-modified proteins (black) compared with that of the total identified proteins (gray) is shown. The number of proteins in each category is expressed as a percentage of the total.

Fig. 5.

Top canonical pathways from mouse brain (black) and heart (gray) represented by DOPA/DQ-modified proteins. The p value is a measure of the likelihood that the association between the set of modified proteins with the given pathway is due to random chance. PI3K, phosphatidylinositol 3-kinase.