Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of limitations of quantitative single-cell protein analysis. To overcome this limitation, we developed SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantified over 3,042 proteins in 1,490 single monocytes and macrophages in ten days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. This gradient correlates to the inflammatory axis of classically and alternatively activated macrophages. Parallel measurements of transcripts by 10x Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus SCoPE2 supports quantification with improved count statistics. The joint distributions of proteins and transcripts allowed exploring regulatory interactions, such as between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Our methodology lays the foundation for quantitative single-cell analysis of proteins by mass-spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.

中文翻译：

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巨噬细胞异质性的单细胞蛋白质组学和转录组学分析

巨噬细胞是具有多种功能和分子表型的先天免疫细胞。由于定量单细胞蛋白质分析的局限性，这种多样性在单细胞蛋白质组学水平上尚未得到充分探索。为了克服这一局限性，我们开发了SCoPE2，它通过引入自动化和小型化的样品制备技术，大大提高了定量准确性和通量，同时降低了成本和动手时间。这些进展使我们能够分析细胞异质性的出现，因为在没有极化细胞因子的情况下，同质单核细胞分化为巨噬细胞样细胞。在10天的仪器时间内，SCoPE2对1,490个单核细胞和巨噬细胞中的3,042种蛋白质进行了定量，这些定量的蛋白质使我们能够按细胞类型区分单细胞。此外，数据揭示了巨噬细胞的蛋白质组状态的连续梯度，表明巨噬细胞的异质性可能在没有极化细胞因子的情况下出现。该梯度与经典的和可替代地活化的巨噬细胞的炎性轴相关。通过10倍基因组学对转录本进行的并行测量表明，我们的测量所采集的蛋白质拷贝数比每个基因的RNA拷贝数高20倍，因此SCoPE2支持定量计数改进的定量分析。蛋白质和转录本的联合分布允许探索调节相互作用，例如抑癌基因p53，其转录本和受p53调控的基因的转录本之间。