Zamboni Nicola, Fendt Sarah-Maria, Rühl Martin, Sauer Uwe
Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
Nat Protoc. 2009;4(6):878-92. doi: 10.1038/nprot.2009.58. Epub 2009 May 21.
Stable isotope, and in particular (13)C-based flux analysis, is the exclusive approach to experimentally quantify the integrated responses of metabolic networks. Here we describe a protocol that is based on growing microbes on (13)C-labeled glucose and subsequent gas chromatography mass spectrometric detection of (13)C-patterns in protein-bound amino acids. Relying on publicly available software packages, we then describe two complementary mathematical approaches to estimate either local ratios of converging fluxes or absolute fluxes through different pathways. As amino acids in cell protein are abundant and stable, this protocol requires a minimum of equipment and analytical expertise. Most other flux methods are variants of the principles presented here. A true alternative is the analytically more demanding dynamic flux analysis that relies on (13)C-pattern in free intracellular metabolites. The presented protocols take 5-10 d, have been used extensively in the past decade and are exemplified here for the central metabolism of Escherichia coli.
稳定同位素，特别是基于¹³C的通量分析，是通过实验定量代谢网络综合反应的唯一方法。在此，我们描述了一种方案，该方案基于在¹³C标记的葡萄糖上培养微生物，并随后通过气相色谱 - 质谱法检测蛋白质结合氨基酸中的¹³C模式。依靠公开可用的软件包，我们接着描述两种互补的数学方法，以估计收敛通量的局部比率或通过不同途径的绝对通量。由于细胞蛋白质中的氨基酸丰富且稳定，该方案需要最少的设备和分析专业知识。大多数其他通量方法都是此处介绍的原理的变体。一种真正的替代方法是分析要求更高的动态通量分析，它依赖于游离细胞内代谢物中的¹³C模式。所介绍的方案需要5 - 10天，在过去十年中已被广泛使用，在此以大肠杆菌的中心代谢为例进行说明。
