Danielson Steven R, Held Jason M, Schilling Birgit, Oo May, Gibson Bradford W, Andersen Julie K
Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, California 94945, USA.
Anal Chem. 2009 Sep 15;81(18):7823-8. doi: 10.1021/ac901176t.
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.
α-突触核蛋白是路易小体的主要成分，路易小体是帕金森病（PD）的主要标志，为蛋白质包涵体。通过针对3-硝基酪氨酸（3NT）的特异性抗体以及质谱分析（MS）确定，路易小体含有高水平的硝化酪氨酸残基。我们开发了一种多反应监测（MRM）质谱方法，用于灵敏地定量特定α-突触核蛋白酪氨酸残基的3NT水平。在一种帕金森病的诱导性转基因细胞模型中，我们发现α-突触核蛋白第39位酪氨酸残基的3NT水平相对于对照增加了9倍，该模型中过表达单胺氧化酶B（MAO-B），模拟了帕金森病的若干特征。通过添加特异性MAO-B抑制剂司来吉兰，可以消除因MAO-B水平升高导致的内源性α-突触核蛋白第39位酪氨酸硝化增加。3NT水平的增加对第39位酪氨酸具有选择性，因为在该蛋白的其他酪氨酸残基（第125位、第133位和第136位酪氨酸）处未检测到3NT水平有显著增加。这是关于帕金森病模型中特定酪氨酸的3NT水平升高的首次报道，也是首次使用MRM质谱法定量目标蛋白特定位点3NT修饰变化的报道。
