Drummond Eleanor, Nayak Shruti, Pires Geoffrey, Ueberheide Beatrix, Wisniewski Thomas
Department of Neurology, Center for Cognitive Neurology, NYU School of Medicine, New York, NY, USA.
Proteomics Laboratory, Division of Advanced Research and Technology, New York University School of Medicine, New York, NY, USA.
Methods Mol Biol. 2018;1723:319-334. doi: 10.1007/978-1-4939-7558-7_18.
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 ~900 proteins in plaques and ~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.
在此，我们描述了一种新方法，该方法可实现对淀粉样斑块和神经原纤维缠结（NFTs）进行局部蛋白质组学分析，而淀粉样斑块和神经原纤维缠结是阿尔茨海默病（AD）的两个病理标志。使用免疫组织化学对淀粉样斑块和NFTs进行可视化，并使用激光捕获显微切割技术从存档的、福尔马林固定石蜡包埋（FFPE）的人体组织样本中进行显微切割。大多数人体组织标本是FFPE；因此，使用这类组织是该技术的一个特别优势。显微切割的组织样本用甲酸溶解、脱石蜡、还原、烷基化、进行蛋白水解消化并脱盐。使用无标记定量液相色谱-质谱法测定斑块和NFTs中所得的蛋白质含量。这使得能够对斑块中约900种蛋白质和NFTs中约500种蛋白质进行无偏且同时的定量分析。这种方法允许进行下游通路和网络分析，从而全面概述在AD神经病理特征中发现的病理性蛋白质积累情况。
