Grankvist Nina, Watrous Jeramie D, Jain Mohit, Nilsson Roland
Cardiovascular Medicine Unit, Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden.
Karolinska University Hospital, Stockholm, Sweden.
Methods Mol Biol. 2020;2088:73-92. doi: 10.1007/978-1-0716-0159-4_5.
The recently developed deep labeling method allows for large-scale profiling of metabolic activities in human cells or tissues using isotope tracing with a highly C enriched culture medium in combination with liquid chromatography-high resolution mass spectrometry. This method generates mass spectrometry data sets where endogenous cellular products can be identified, and active pathways can be determined from observed C mass isotopomers of the various metabolites measured. Here we describe in detail the experimental procedures for deep labeling experiments in cultured mammalian cells, including synthesis of the deep labeling medium, experimental considerations for cell culture, metabolite extractions and sample preparation, and liquid chromatography-mass spectrometry. We also outline a workflow for the downstream data analysis using publicly available software.
最近开发的深度标记方法允许使用富含碳的培养基进行同位素示踪，并结合液相色谱-高分辨率质谱，对人类细胞或组织中的代谢活动进行大规模分析。该方法生成质谱数据集，可识别内源性细胞产物，并根据所测量的各种代谢物的碳质量同位素异构体确定活跃途径。在这里，我们详细描述了在培养的哺乳动物细胞中进行深度标记实验的实验程序，包括深度标记培养基的合成、细胞培养的实验注意事项、代谢物提取和样品制备以及液相色谱-质谱分析。我们还概述了使用公开可用软件进行下游数据分析的工作流程。
