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首页> 外文期刊>Molecular & cellular proteomics: MCP >Streamlined Protocol for Deep Proteomic Profiling of FAC-sorted Cells and Its Application to Freshly Isolated Murine Immune Cells
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Streamlined Protocol for Deep Proteomic Profiling of FAC-sorted Cells and Its Application to Freshly Isolated Murine Immune Cells

机译：流线型方案，用于区分区分选细胞的深层蛋白质组学分析及其在新鲜孤立的鼠免疫细胞中的应用

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Proteomic profiling describes the molecular landscape of proteins in cells immediately available to sense, transduce, and enact the appropriate responses to extracellular queues. Transcriptional profiling has proven invaluable to our understanding of cellular responses; however, insights may be lost as mounting evidence suggests transcript levels only moderately correlate with protein levels in steady state cells. Mass spectrometry-based quantitative proteomics is a well-suited and widely used analytical tool for studying global protein abundances. Typical proteomic workflows are often limited by the amount of sample input that is required for deep and quantitative proteome profiling. This is especially true if the cells of interest need to be purified by fluorescence-activated cell sorting (FACS) and one wants to avoid ex vivo culturing. To address this need, we developed an easy to implement, streamlined workflow that enables quantitative proteome profiling from roughly 2 mu g of protein input per experimental condition. Utilizing a combination of facile cell collection from cell sorting, solid-state isobaric labeling and multiplexing of peptides, and small-scale fractionation, we profiled the proteomes of 12 freshly isolated, primary murine immune cell types. Analyzing half of the 3e5 cells collected per cell type, we quantified over 7000 proteins across 12 key immune cell populations directly from their resident tissues. We show that low input proteomics is precise, and the data generated accurately reflects many aspects of known immunology, while expanding the list of cell-type specific proteins across the cell types profiled. The low input proteomics methods we developed are readily adaptable and broadly applicable to any cell or sample types and should enable proteome profiling in systems previously unattainable. Molecular & Cellular Proteomics 18: 995-1009, 2019. DOI: 10.1074/mcp.RA118.001259.
机译：蛋白质组学分析描述了可感知，转诱导的细胞中蛋白质的分子景观，并对细胞外队列制定适当的反应。转录分析已经证明我们对细胞反应的理解非常宝贵;然而，由于安装证据表明转录物水平同时与稳态细胞中的蛋白质水平，洞察力可能丢失。基于质谱的定量蛋白质组学是一种良好且广泛使用的分析工具，用于研究全球蛋白质丰富。典型的蛋白质组学工作流程通常受到深度和定量蛋白质组分析所需的样品输入量的限制。如果需要通过荧光激活的细胞分选（FACS）纯化感兴趣的细胞，并且想要避免离体培养，则这尤其如此。为了解决这种需求，我们开发了一种易于实现的简化流线型，使定量蛋白质组分析从大约2μg的每种实验条件输入。利用来自细胞分选的容易细胞收集，固态等异质标记和多重肽的多路复用，以及小规模分馏，我们突出了12次新鲜的初级鼠免疫细胞类型的蛋白质蛋白质。分析每种细胞类型收集的3E5细胞的一半，我们直接从其驻留组织中量化超过7000个蛋白质。我们表明，低输入蛋白质组学是精确的，并且精确地产生的数据反映了已知免疫学的许多方面，同时扩展细胞型特异性蛋白质的列表，这些蛋白质跨越细胞类型。我们开发的低输入蛋白质组学方法是易于适应的并且广泛适用于任何细胞或样品类型，并应使蛋白质组分布在以前无法实现的系统中。分子和细胞蛋白质组学18：995-1009,2017。DOI：10.1074 / MCP.RA118.001259。

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