Quantifying Intermediary Metabolism and Lipogenesis in Cultured Mammalian Cells Using Stable Isotope Tracing and Mass Spectrometry
Quantifying Intermediary Metabolism and Lipogenesis in Cultured Mammalian Cells Using Stable Isotope Tracing and Mass Spectrometry
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DOI：
10.1007/978-1-4939-9236-2_14
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发表时间：
2019-01-01
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影响因子：
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通讯作者：
Metallo, Christian M.
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文献类型：
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作者：
Cordes, Thekla;Metallo, Christian M.
新陈代谢在几乎所有疾病中都起着核心作用，包括糖尿病、癌症和神经变性。需要进行详细的分析，以确定在特定疾病或生物扰动的背景下失调的特定代谢途径。对代谢产物浓度的测量可以提供对改变的途径活性或酶功能的一些见解，但由于大多数生物化学物质是由细胞和组织内不同途径的不同酶代谢的，这些方法在一定程度上是有限的。通过将代谢示踪剂应用于生物系统，人们可以根据所使用的示踪剂和所测量的分析物来可视化特定路径的信息。为此，稳定同位素示踪剂和质谱学正在成为研究培养的哺乳动物细胞和其他系统中代谢途径和通量的重要工具。在这里，我们描述了一个详细的工作流程，用于量化哺乳动物细胞培养中的代谢过程，使用稳定同位素和气相色谱-质谱仪(GC-MS)。作为一个案例研究，我们将C-13同位素标记的葡萄糖和谷氨酰胺应用于癌细胞系，以量化TCA代谢和脂肪生成的底物利用。还提供了解释数据的指南和应用于其他蜂窝系统的考虑因素。最终，这种方法提供了一种稳健和精确的方法来量化代谢物库中的稳定同位素标记，可以应用于不同的生物系统。
Metabolism plays a central role in virtually all diseases, including diabetes, cancer, and neurodegeneration. Detailed analysis is required to identify the specific metabolic pathways dysregulated in the context of a given disease or biological perturbation. Measurement of metabolite concentrations can provide some insights into altered pathway activity or enzyme function, but since most biochemicals are metabolized by various enzymes in distinct pathways within cells and tissues, these approaches are somewhat limited. By applying metabolic tracers to a biological system, one can visualize pathway-specific information depending on the tracer used and analytes measured. To this end, stable isotope tracers and mass spectrometry are emerging as important tools for the examination of metabolic pathways and fluxes in cultured mammalian cells and other systems. Here, we describe a detailed workflow for quantifying metabolic processes in mammalian cell cultures using stable isotopes and gas chromatography coupled to mass spectrometry (GC-MS). As a case study, we apply C-13 isotopic labeled glucose and glutamine to a cancer cell line to quantify substrate utilization for TCA metabolism and lipogenesis. Guidelines are also provided for interpretation of data and considerations for application to other cell systems. Ultimately, this approach provides a robust and precise method for quantifying stable isotope labeling in metabolite pools that can be applied to diverse biological systems.