Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal.
J Neurochem. 2013 Sep;126(5):673-84. doi: 10.1111/jnc.12251. Epub 2013 Apr 25.
α-Synuclein (α-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of α-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α-syn PTMs. Here, by taking advantage of α-syn thermostability, we applied a method to achieve high enrichment of soluble α-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies.
α-突触核蛋白（α-syn）是路易体的主要成分，路易体是帕金森病和其他突触核蛋白病的病理标志。α-syn 的翻译后修饰（PTMs）的特征，被认为会干扰其聚集倾向和细胞信号转导，但受到从细胞和组织中提取内源性蛋白质的方法以及针对 α-syn PTM 的抗体的可用性的限制。在这里，我们利用 α-syn 的热稳定性，应用一种方法从培养的细胞和脑组织中实现可溶性 α-syn 的高富集，然后进行蛋白质组学分析。使用这种方法，我们在包括帕金森病转基因小鼠模型在内的各种模型系统中获得了 98%的 α-syn 序列覆盖度，并通过鉴定先前描述的 PTM 如磷酸化和 N 端乙酰化来验证该策略。我们的发现表明，该程序克服了现有技术限制，可以用于促进 α-syn PTM 的系统研究，从而阐明其在生理和病理条件下的作用。最终，这种方法可能能够开发新的生物标志物和治疗干预突触核蛋白病的策略。
