系统和功能生物学中代谢物和蛋白质的单细胞质谱分析。

Erika P Portero, Leena Pade, Jie Li, Sam B Choi, Peter Nemes
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引用次数: 0

摘要

分子组成与细胞功能错综复杂地交织在一起,阐明这种关系对于理解生命过程和开发下一代治疗方法至关重要。毛细管电泳(CE)和液相色谱(LC)质谱(MS)的技术创新通过允许高特异性的无偏检测和分子定量,提供了以前无法获得的细胞生物化学见解。本章介绍了我们经过验证的方案,将超灵敏质谱与细胞、发育和神经生物学的经典工具相结合,以评估重要生物分子的生物学功能。我们使用CE和LC MS测量脊索动物胚胎和哺乳动物神经元中单个细胞或有限群体组织中的数百种代谢物和数千种蛋白质,揭示鉴定细胞之间的分子异质性。通过显微注射和光学显微镜的结合,我们展示了细胞谱系追踪,并测试了失调分子在青蛙胚胎(Xenopus laevis)细胞异质性和组织规格的形成和维持中的作用。电生理学将我们的工作流程扩展到表征哺乳动物脑组织部分的神经元活动。从这些研究中获得的信息相互加强了化学和生物学,并突出了跨学科研究对推进基础知识和转化应用的重要性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Single-Cell Mass Spectrometry of Metabolites and Proteins for Systems and Functional Biology.

Molecular composition is intricately intertwined with cellular function, and elucidation of this relationship is essential for understanding life processes and developing next-generational therapeutics. Technological innovations in capillary electrophoresis (CE) and liquid chromatography (LC) mass spectrometry (MS) provide previously unavailable insights into cellular biochemistry by allowing for the unbiased detection and quantification of molecules with high specificity. This chapter presents our validated protocols integrating ultrasensitive MS with classical tools of cell, developmental, and neurobiology to assess the biological function of important biomolecules. We use CE and LC MS to measure hundreds of metabolites and thousands of proteins in single cells or limited populations of tissues in chordate embryos and mammalian neurons, revealing molecular heterogeneity between identified cells. By pairing microinjection and optical microscopy, we demonstrate cell lineage tracing and testing the roles the dysregulated molecules play in the formation and maintenance of cell heterogeneity and tissue specification in frog embryos (Xenopus laevis). Electrophysiology extends our workflows to characterizing neuronal activity in sections of mammalian brain tissues. The information obtained from these studies mutually strengthen chemistry and biology and highlight the importance of interdisciplinary research to advance basic knowledge and translational applications forward.

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