Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2.
Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2.
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DOI：
10.1186/s13059-021-02267-5
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发表时间：
2021-01-27
期刊：
影响因子：
12.3
通讯作者：
Slavov N
中科院分区：
文献类型：
--
作者：
Specht H;Emmott E;Petelski AA;Huffman RG;Perlman DH;Serra M;Kharchenko P;Koller A;Slavov N
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水平上很大程度上未被探索。为了克服这一限制，我们开发了 SCoPE2，它通过引入自动化和小型化样品制备，显着提高了定量精度和通量，同时降低了成本和操作时间。这些进展使我们能够分析细胞异质性的出现，因为同质单核细胞在没有极化细胞因子的情况下分化为巨噬细胞样细胞。 SCoPE2 在 10 天的仪器时间内对 1490 个单个单核细胞和巨噬细胞中的超过 3042 个蛋白质进行了定量，定量的蛋白质使我们能够按细胞类型辨别单个细胞。此外，数据揭示了巨噬细胞蛋白质组状态的连续梯度，表明巨噬细胞异质性可能在缺乏极化细胞因子的情况下出现。 10× Genomics 对转录本的并行测量表明，我们的测量样本中每个基因的蛋白质拷贝数是 RNA 拷贝数的 20 倍，因此，SCoPE2 支持通过改进的计数统计进行量化。这使得探索调控相互作用成为可能，例如肿瘤抑制因子 p53、其转录物和受 p53 调控的基因转录物之间的相互作用。即使在同质环境中，巨噬细胞蛋白质组也是异质的。这种异质性与经典和替代激活的巨噬细胞的炎症轴相关。我们的方法为通过质谱法对蛋白质进行自动化和定量单细胞分析奠定了基础，并证明了从单细胞变异性推断转录和转录后调控的潜力。在线版本包含可在 10.1186/s13059-021-02267-5 获取的补充材料。
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 the limitations of quantitative single-cell protein analysis. To overcome this limitation, we develop 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 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 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. Parallel measurements of transcripts by 10× 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. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and 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. The online version contains supplementary material available at 10.1186/s13059-021-02267-5.
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