Development of a Sensitive, Scalable Method for Spatial, Cell-Type-Resolved Proteomics of the Human Brain
Development of a Sensitive, Scalable Method for Spatial, Cell-Type-Resolved Proteomics of the Human Brain
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DOI：
10.1021/acs.jproteome.8b00981
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发表时间：
2019-04-01
影响因子：
4.4
通讯作者：
Fischer, Roman
中科院分区：
文献类型：
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作者：
Davis, Simon;Scott, Connor;Fischer, Roman
虽然几乎全面的蛋白质组覆盖范围可以实现从散装组织或培养细胞，数据通常缺乏空间分辨率。因此，基于组织的蛋白质组学在多种细胞类型和/或定位中平均蛋白质丰度。由于蛋白质组学平台缺乏进行深入的单细胞蛋白质组研究以解析组织内的空间或细胞类型依赖性蛋白质表达梯度的灵敏度和通量，蛋白质组学分析已与分选技术相结合以富集某些细胞群体。然而，空间分辨率和上下文在细胞分选后丢失。在这里，我们报告了一个优化的方法，从死后人脑分离的神经元的蛋白质组学分析激光捕获显微切割（LCM）。我们测试了样品收集方法、裂解缓冲液和消化方法的组合，以最大限度地提高鉴定数量和定量性能，从60 000 μ m（2）10 μ m厚的小脑分子层中鉴定出1500种蛋白质，具有良好的重现性。为了证明我们的工作流程解析人脑组织中细胞类型特异性蛋白质组的能力，我们分离了一组单独的贝茨和浦肯野细胞。这两种神经元细胞类型都参与运动协调，并被发现表达高度特异性的蛋白质组，深度为2800至3600个蛋白质。
While nearly comprehensive proteome coverage can be achieved from bulk tissue or cultured cells, the data usually lacks spatial resolution. As a result, tissue based proteomics averages protein abundance across multiple cell types and/or localizations. With proteomics platforms lacking sensitivity and throughput to undertake deep single-cell proteome studies in order to resolve spatial or cell type dependent protein expression gradients within tissue, proteome analysis has been combined with sorting techniques to enrich for certain cell populations. However, the spatial resolution and context is lost after cell sorting. Here, we report an optimized method for the proteomic analysis of neurons isolated from post-mortem human brain by laser capture microdissection (LCM). We tested combinations of sample collection methods, lysis buffers and digestion methods to maximize the number of identifications and quantitative performance, identifying 1500 proteins from 60 000 mu m(2) of 10 mu m thick cerebellar molecular layer with excellent reproducibility. To demonstrate the ability of our workflow to resolve cell type specific proteomes within human brain tissue, we isolated sets of individual Betz and Purkinje cells. Both neuronal cell types are involved in motor coordination and were found to express highly specific proteomes to a depth of 2800 to 3600 proteins.