Center of Innovative Research, Banyan Biomarkers, Inc., Alachua, FL, USA.
Electrophoresis. 2011 Jun;32(13):1692-705. doi: 10.1002/elps.201100133.
Protein tyrosine nitration is a post-translational modification commonly used as a marker of cellular oxidative stress associated with numerous pathophysiological conditions. We focused on ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1) and glyceraldehyde-3-phosphate (GAPDH) which are high-abundant brain proteins that have been identified to be highly susceptible to oxidative modification. Both UCH-L1 and GAPDH have been linked to the pathogenesis of Alzheimer's and Parkinson's disease, however specific nitration sites have not been elucidated. Identification of specific nitration sites and quantitation of endogenous nitrated proteins are important in correlating this modification to disease pathology. In this study, purified UCH-L1 and GAPDH were nitrated in vitro with peroxynitrite and the presence of nitrated proteins was confirmed by anti-3-nitrotyrosine Western blots. Data-dependent LC-MS/MS analysis identified several distinct tyrosine nitration sites in UCH-L1 (Tyr-80) and GAPDH (Tyr-47, Tyr-92, and Tyr-312). Subsequent validation with synthetic peptides was conducted for selected nitropeptides. An LC-MS/MS method was developed for semi-quantitative determination of the synthetic nitropeptides: KGQEVSPKVY() (UCH-L1) and mFQY() DSTHGKF (GAPDH). The nitropeptides were detectable in the mid-attomole range and the peak area response was linear over three orders of magnitude. Targeted analysis of endogenous UCH-L1 and GAPDH nitration was then conducted in an in vivo second-hand smoke rat model to evaluate the utility of this approach.
蛋白质酪氨酸硝化是一种常见的翻译后修饰，通常用作与许多病理生理状况相关的细胞氧化应激的标志物。我们专注于泛素羧基末端水解酶-L1（UCH-L1）和甘油醛-3-磷酸（GAPDH），它们是大脑中高丰度的蛋白质，已被确定易受氧化修饰。UCH-L1 和 GAPDH 都与阿尔茨海默病和帕金森病的发病机制有关，但特定的硝化位点尚未阐明。鉴定特定的硝化位点和定量内源性硝化蛋白对于将这种修饰与疾病病理学相关联非常重要。在这项研究中，纯化的 UCH-L1 和 GAPDH 在体外用过氧亚硝酸盐硝化，并用抗 3-硝基酪氨酸 Western blot 确认硝化蛋白的存在。依赖数据的 LC-MS/MS 分析鉴定了 UCH-L1（Tyr-80）和 GAPDH（Tyr-47、Tyr-92 和 Tyr-312）中的几个不同的酪氨酸硝化位点。随后对选定的硝化肽进行了合成肽的后续验证。开发了一种 LC-MS/MS 方法用于半定量测定合成硝化肽：KGQEVSPKVY()（UCH-L1）和 mFQY() DSTHGKF（GAPDH）。这些硝化肽在中 attomole 范围内可检测到，并且峰面积响应在三个数量级范围内呈线性。然后在体内二手烟大鼠模型中对内源性 UCH-L1 和 GAPDH 硝化进行了靶向分析，以评估这种方法的实用性。
