Preferentially increased nitration of alpha-synuclein at tyrosine-39 in a cellular oxidative model of Parkinson's disease

Abstract

Alpha-synuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinson's disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3-nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alpha-synuclein tyrosine residues. We found a 9-fold increase (relative to controls) in levels of 3NT at Tyr-39 of alpha-synuclein in an inducible transgenic cellular model of Parkinson's disease in which monoamine oxidase B (MAO-B) is overexpressed and which emulates several features of PD. Increased nitration of Tyr-39 on endogenous alpha-synuclein via elevations in MAO-B levels could be abrogated by the addition of deprenyl, a specific MAO-B inhibitor. The increased levels of 3NT was selective for Tyr-39 as no significant increases in 3NT levels were detected at other tyrosine residues present in the protein (Tyr-125, Tyr-133, and Tyr-136). This is the first report of increased 3NT levels of a specific tyrosine in a PD model and the first use of MRM mass spectrometry to quantify changes in 3NT modifications at specific sites within a target protein.

Figure 1

Diagram depicting summarized strategy to identify and quantify PTMs (3NT) on low abundant peptides, starting with in vitro nitrated (tetranitromethane) recombinant α-synuclein which was subsequently digested and analyzed by MS/MS to identify 3NT modified peptides using rigorous criteria described in the Results section. Optimized MRM transitions were then used to quantify 3NT modified and unmodified peptides present on endogenous α-synuclein immunoprecipitated (IP) from a cellular model of PD using the equation listed in the final step titled MRM analysis (TIC = Total Ion Chromatogram).

Figure 2

Identification of α-synuclein peptide E-35-GVLY^NO2VGSKTK-45 containing 3NT modification at Tyr-39. Human α-synuclein was in vitro nitrated, proteolytically digested with Asp-N and then analyzed by MS/MS. A) MS/MS spectra of unmodified human α-synuclein peptide (Asp-N digested) containing Tyr-39, E-35-GVLYVGSKTK-45 at m/z 394.2, (M=1179.6 Da). B) Corresponding MS/MS spectra of the peptide containing 3NT, E-35-GVLY^NO2VGSKTK-45 (Y^NO2 = 3NT) at m/z 409.2 (M=1224.5 Da). Note the low mass immonium (IMM) ions (+NH₂=CH-R), for Tyr and 3NT at m/z 136.1 and 181.1, respectively.

Figure 3

Chromatographic retention time shift of 3NT modified peptide. MRM total ion chromatogram of unmodified and 3NT modified peptide (Asp-N digest) containing Tyr-39 showing retention time delay of ~2 min for the nitrated peptide relative to the unmodified form. Four MRM transitions were used each for the unmodified and 3NT modified peptides, all of which are listed in Table S-2 (Supporting Information).

Figure 4

Increased 3NT levels at Tyr-39 of endogenous α-synuclein due to MAO-B overexpression in dopaminergic PC12 cells as determined by MRM (4000 QTRAP). A 9-fold increase of 3NT levels at Tyr-39 was observed in PC12 cells overexpressing MAO-B relative to control levels. This increase was abrogated by the addition of the MAO-B inhibitor deprenyl. As a positive control, cells were treated with 150 μM of H₂O₂ for 24 hours which also exhibited a large increase (6-fold) in 3NT levels as compared to control cells. Quantitation was done using the MRM transitions (Q1/Q3): unmodified 394.2/520.3 and 3NT modified 409.2/520.3. Error bars are the standard deviation of 3 biological replicates analyzed in singlicate using MRM transitions for unmodified and 3NT modified peptides.