Montreal Clinical Research Institute (IRCM), 110 Avenue Des Pins Ouest, Montreal, QC H2W 1R7, Canada; Department of Biochemistry and Molecular Medicine, University of Montreal, C.P. 6128, Succursale Centre-ville, Montreal, QC H3C 3J7, Canada.
Montreal Clinical Research Institute (IRCM), 110 Avenue Des Pins Ouest, Montreal, QC H2W 1R7, Canada.
Methods. 2022 Dec;208:9-18. doi: 10.1016/j.ymeth.2022.09.009. Epub 2022 Oct 10.
Cellular heterogeneity is fundamental to both developmental differentiation and disease establishment. Recent advances in high-throughput single-cell technology have been rapidly revolutionizing the resolution of our understanding of development and disease. However, while the study of single-cell transcriptomes is easily accessible, the analysis of single-cell proteomes is still in its infancy. In this study, we describe simultaneous profiling of multiple regulatory proteins at a single-cell level using mass cytometry or cytometry by time of flight. We develop mass cytometry reagents to study key transcription factors, signaling proteins and chromatin modifiers that regulate mouse embryonic stem cells. Our data reveal that the protein level of stem cell regulators significantly varies and that cell signaling pathways are extensively cross-activated across defined culture conditions of embryonic stem cells. In addition, the mass cytometry data enabled us to identify distinct multiple cell states of embryonic stem cells and determine their variation across culture conditions. We discuss the mass cytometry method, our results of the multi-protein analysis in embryonic stem cells and potential future perspectives for single-cell protein analysis.
细胞异质性是发育分化和疾病发生的基础。高通量单细胞技术的最新进展正在迅速改变我们对发育和疾病的理解。然而，尽管单细胞转录组的研究易于实现，但单细胞蛋白质组的分析仍处于起步阶段。在这项研究中，我们使用质谱流式细胞术或飞行时间流式细胞术描述了在单细胞水平上同时对多种调节蛋白进行分析。我们开发了质谱流式细胞术试剂来研究调控小鼠胚胎干细胞的关键转录因子、信号蛋白和染色质修饰剂。我们的数据表明，干细胞调控蛋白的水平存在显著差异，并且细胞信号通路在胚胎干细胞的明确定义的培养条件下广泛交叉激活。此外，质谱流式细胞术数据使我们能够识别胚胎干细胞的不同多细胞状态，并确定它们在培养条件下的变化。我们讨论了质谱流式细胞术方法、我们在胚胎干细胞中的多蛋白分析结果以及单细胞蛋白质分析的潜在未来展望。
