Isolation of Amyloid Plaques and Neurofibrillary Tangles from Archived Alzheimer's Disease Tissue Using Laser-Capture Microdissection for Downstream Proteomics
Isolation of Amyloid Plaques and Neurofibrillary Tangles from Archived Alzheimer's Disease Tissue Using Laser-Capture Microdissection for Downstream Proteomics
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DOI：
10.1007/978-1-4939-7558-7_18
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发表时间：
2018-01-01
期刊：
影响因子：
--
通讯作者：
Wisniewski, Thomas
中科院分区：
文献类型：
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作者：
Drummond, Eleanor;Nayak, Shruti;Wisniewski, Thomas
在这里，我们描述了一种新的方法，它可以对阿尔茨海默病(AD)的两个病理特征--淀粉样斑块和神经原纤维缠结(NFT)进行局部蛋白质组学研究。淀粉样斑块和NFT使用免疫组织化学可视化，并使用激光捕获显微解剖从存档的福尔马林固定石蜡包埋(FFPE)的人类组织样本中进行显微解剖。大多数人体组织标本是FFPE；因此，使用这种类型的组织是这项技术的一个特别优势。显微解剖的组织样品用甲酸溶解，去烷基、还原、烷基化、蛋白水解性消化和脱盐。用无标记定量LC-MS测定斑块和NFT的蛋白质含量。这使得斑块中类似的900个蛋白质和非傅立叶变换中的类似的500个蛋白质被无偏见地同时定量。这种方法允许下游通路和网络分析，从而提供了在AD的神经病理特征中发现的病理性蛋白积聚的全面概述。
Here, we describe a new method that allows localized proteomics of amyloid plaques and neurofibrillary tangles (NFTs), which are the two pathological hallmarks of Alzheimer's disease (AD). Amyloid plaques and NFTs are visualized using immunohistochemistry and microdissected from archived, formalin-fixed paraffin-embedded (FFPE) human tissue samples using laser-capture microdissection. The majority of human tissue specimens are FFPE; hence the use of this type of tissue is a particular advantage of this technique. Microdissected tissue samples are solubilized with formic acid and deparaffinized, reduced, alkylated, proteolytically digested, and desalted. The resulting protein content of plaques and NFTs is determined using label-free quantitative LC-MS. This results in the unbiased and simultaneous quantification of similar to 900 proteins in plaques and similar to 500 proteins in NFTs. This approach permits downstream pathway and network analysis, hence providing a comprehensive overview of pathological protein accumulation found in neuropathological features in AD.