Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain
Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain
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DOI：
10.3791/55835
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发表时间：
2017-10-01
影响因子：
1.2
通讯作者：
Seyfried, Nicholas T.
中科院分区：
文献类型：
--
作者：
Diner, Ian;Tram Nguyen;Seyfried, Nicholas T.
在这项研究中，我们描述了一个简短的单步分馏协议的富集洗涤剂不溶性蛋白质聚集体从人死后的大脑。离子去污剂N-月桂基-肌氨酸（sarkosyl）有效地溶解脑组织中天然折叠的蛋白质，允许富集来自广泛的神经变性蛋白质病（例如阿尔茨海默病（AD）、帕金森病和肌萎缩性侧索硬化症以及朊病毒病）的去污剂不溶性蛋白质聚集体。将人对照和AD死后脑组织均质化，并在肌氨酰存在下通过超离心沉淀，以富集洗涤剂不溶性蛋白质聚集体，包括病理性磷酸化tau，AD中神经元缠结的核心组分。蛋白质印迹证明了聚集的磷酸化tau蛋白和洗涤剂可溶性蛋白，早期内体抗原1（EEA 1）在对照和AD脑中的溶解度不同。蛋白质组学分析还显示，与对照组相比，AD脑的肌氨酰不溶性组分中β-淀粉样蛋白（A β）、tau、snRNP 70（U1- 70 K）和载脂蛋白E（APOE）富集，这与先前的组织分级策略一致。因此，这种简单的富集方案是理想的广泛的实验应用，从蛋白质印迹和功能蛋白质共聚集测定到基于质谱的蛋白质组学。
In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer's disease (AD), Parkinson's disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of beta-amyloid (A beta), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.