Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.
Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.
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DOI：
10.3791/63816
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发表时间：
2022-04-28
期刊：
影响因子：
--
通讯作者：
Savas JN
中科院分区：
文献类型：
--
作者：
Upadhyay A;Vassar RJ;Savas JN
蛋白质纤维包涵体是多种神经退行性疾病的关键病理标志。在阿尔茨海默病（AD）的早期阶段，淀粉样蛋白-β肽在细胞外空间中形成原纤维，其充当逐渐生长并成熟为大的淀粉样蛋白斑块的种子。尽管有这些基本的了解，目前对淀粉样蛋白原纤维结构、组成和脑中沉积模式的了解仍然有限。一个主要的障碍是无法从脑提取物中分离高度纯化的淀粉样纤维。亲和纯化和基于激光捕获显微切割的方法先前已用于分离淀粉样蛋白，但受限于可回收的少量材料。这种新的，强大的协议描述了淀粉样蛋白斑块核心的生化纯化，使用十二烷基硫酸钠（SDS）增溶与蔗糖密度梯度超离心和超声处理，并产生高纯度的原纤维从AD患者和AD模型脑组织。基于质谱（MS）的纯化材料的自下而上的蛋白质组学分析代表了一个强大的战略，以确定几乎所有的主要蛋白质成分的淀粉样蛋白原纤维。先前对淀粉样冠蛋白的蛋白质组学研究已经揭示了一个意想不到的大的和功能多样的蛋白质集合。值得注意的是，在改进纯化策略后，共纯化蛋白质的数量减少了10倍以上，表明分离的SDS不溶性材料的高纯度。负染色和免疫金电子显微镜检查允许确认这些制剂的纯度。需要进一步的研究，以了解有助于这些蛋白质沉积成淀粉样包涵体的空间和生物学属性。总的来说，这种分析策略很好地定位于增加对淀粉样蛋白生物学的理解。这种基于质谱的蛋白质组学分析的生化纯化方法有助于淀粉样蛋白原纤维核心的稳健表征，这可能会加速预防阿尔茨海默病的靶点的鉴定。
Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer’s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology. This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer’s disease.
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