Localized Proteomics of Individual Neurons Isolated from Formalin-Fixed, Paraffin-Embedded Tissue Sections Using Laser Capture Microdissection
Localized Proteomics of Individual Neurons Isolated from Formalin-Fixed, Paraffin-Embedded Tissue Sections Using Laser Capture Microdissection
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DOI：
10.1007/978-1-4939-7119-0_18
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发表时间：
2017-01-01
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影响因子：
--
通讯作者：
Wisniewski, Thomas
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文献类型：
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作者：
Drummond, Eleanor;Nayak, Shruti;Wisniewski, Thomas
特定细胞群体的局部蛋白质组学将极大地帮助理解神经退行性疾病的复杂病因。与传统蛋白质检测技术相比，蛋白质组学的使用具有相当大的优势，并且可能对研究神经退行性疾病特别有用，因为特定神经元群体的选择性脆弱性是这些疾病的定义特征。发病机制可能涉及多种蛋白质之间的复杂相互作用。因此，优选能够同时定量细胞群中所有蛋白质的技术。方法，使蛋白质组学从存档福尔马林固定，石蜡包埋（FFPE）组织分离的细胞是特别有利于未来的研究，因为大FFPE组织库。在这里，我们描述了我们的协议，本地化的蛋白质组学的神经元显微解剖存档FFPE阿尔茨海默氏病的脑组织。通过用甲酚紫染色使神经元可视化，并使用激光捕获显微切割（LCM）分离。收集1.5 mm（2）的总组织面积（约12，000个神经元）为定量质谱法提供了足够的材料。将切除的组织直接脱蜡、还原、烷基化、蛋白水解消化、脱盐并使用LC-MS进行分析。
Localized proteomics of specific cell populations will greatly aid understanding of the complex etiology of neurodegenerative diseases. The use of proteomics offers considerable advantages over traditional protein detection techniques and is likely to be particularly useful for studying neurodegenerative diseases, as selective vulnerability of specific neuron populations is a defining characteristic of these diseases. Pathogenesis is likely to involve complex interactions between multiple proteins. Therefore, a technique that can quantify all proteins in a cell population at the same time is preferred. Methodology that enables proteomics on cells isolated from archived formalin-fixed, paraffin-embedded (FFPE) tissue is of particular benefit for future studies because of the large FFPE tissue repositories. Here, we describe our protocol for localized proteomics of neurons microdissected from archived FFPE Alzheimer's disease brain tissue. Neurons are visualized by staining with cresyl violet and isolated using laser capture microdissection (LCM). Collection of 1.5 mm(2) total tissue area (approximately 12,000 neurons) provides enough material for quantitative mass spectrometry. The excised tissue is directly deparaffinized, reduced, alkylated, proteolytically digested, desalted, and analyzed using LC-MS.