Preferentially Increased Nitration of α-Synuclein at Tyrosine-39 in a Cellular Oxidative Model of Parkinson’s Disease

Abstract

Abstractα-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 α-synuclein tyrosine residues. We found a nine-fold increase (relative to controls) in levels of 3NT at Tyr-39 of α-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 α-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 . 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.

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

“…This selective mode of data acquisition permits the sensitive detection and identification of peptide abundance over a wide range of protein abundance within complex sample mixtures (118,119). SRM-MS has already been employed for the quantification of nitrated ␣-syn and proteins in biological or clinical samples and for targeting PTMs for which adequate antibodies often do not exist (102). SRM-MS could serve as a valuable analytical tool for the validation of ␣-syn as a biomarker.…”

“…Danielson et al introduced combined proteolysis with trypsin and Asp-N to increase the sequence coverage (Figs. 4 and 5D), which enabled the successful identification of all four 3NT residues, of which one is found in the N-terminal region (Y39) and three (Y125, Y133, and Y136) are located within the C-terminal region of ␣-syn (102).…”

Proteomics quantification of protein nitration

“…Nitrated peptides upon gas-phase fragmentation result in a peak at m/z 181.1, which is the immonium ion of nitrotyrosine (Danielson et al 2009 ). Selected (SRM) or multiple reaction monitoring (MRM) allows selective detection of fragments from nitrated peptides by scanning the Q3 of a triple quadrupole MS in a collision-induced dissociation experiment.…”

“…Neurodegenerative disorders such as PD ( Danielson et al 2009 ), Down syndrome (Jovanovic et al 1998 ), Huntington ' s disease (Browne et al 1999 ), and amyotrophic lateral sclerosis (Abe et al 1995 , Cookson andShaw 1999 ) are also affected by protein nitration ( Horiguchi et al 2003 , Butterfield et al accepted for publication). Similarly, elevated levels of nitration are observed in cardiomyopathy, cardiovascular disease, atherosclerosis (Leeuwenburgh et al 1997, Turko and Murad 2002, Ungvari et al 2005, Peluffo and Radi 2007, Upmacis 2008, Lee et al 2009a, Surmeli et al 2010, arthritis (Sandhu et al 2003 ), diabetes (Turko et al 2003 ), cancer (Azad et al 2008 ), and aging (Pacher et al 2007, Marshall et al 2013.…”

Applications of targeted proteomics in systems biology and translational medicine

“…The importance of multisite protein phosphorylation and the value of quantifying it by targeted MS were recently shown for the connexin family of proteins and are described in a review by Chen and coworkers 61. Danielson and coworkers used antibodies specific for 3‐nitrotyrosine to quantify the levels of this modification in alpha‐synuclein residues 62. Held and coworkers developed a new method for studying oxidation in response to reactive oxygen species, termed oxMRM 63.…”