Studies of the topology, functioning, and regulation of metabolic systems are based on two main types of information that can be measured by mass spectrometry: the (absolute or relative) concentration of metabolites and their isotope incorporation in 13C-labeling experiments. These data are currently obtained from two independent experiments because the 13C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the 13C-mass fractions from which its 13C-isotopologue distribution (CID) is quantified. Here, we developed a generic method with a dedicated processing workflow to obtain these two sets of information simultaneously in a unique sample collected from a single cultivation, thereby reducing by a factor of 2 both the number of cultivations to perform and the number of samples to collect, prepare, and analyze. The proposed approach is based on an IS labeled with other isotope(s) that can be resolved from the 13C-mass fractions of interest. As proof-of-principle, we analyzed amino acids using a doubly labeled 15N13C-cell extract as IS. Extensive evaluation of the proposed approach shows a similar accuracy and precision compared to state-of-the-art approaches. We demonstrate the value of this approach by investigating the dynamic response of amino acids metabolism in mammalian cells upon activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a key component of the unfolded protein response. Integration of metabolite concentrations and isotopic profiles reveals a reduced de novo biosynthesis of amino acids upon PERK activation. The proposed approach is generic and can be applied to other (micro)organisms, analytical platforms, isotopic tracers, or classes of metabolites.

中文翻译：

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通过质谱同时测量代谢物浓度和同位素掺入。

对代谢系统的拓扑结构，功能和调控的研究基于可以通过质谱法测量的两种主要信息：代谢物的（绝对或相对）浓度及其在13C标记实验中的同位素掺入。这些数据当前是从两个独立的实验中获得的，因为用于确定给定代谢物浓度的13C标记内标（IS）与13C质量分数重叠，从中定量了13C同位素异构体分布（CID）。在这里，我们开发了一种通用方法，该方法具有专用的处理工作流程，可以在从一次栽培中收集的唯一样品中同时获取这两套信息，从而将要进行的栽培数量和要进行的样品数量减少了2倍收集，准备，和分析。所提出的方法基于标有其他同位素的IS，可以从感兴趣的13C质量分数中分离出该IS。作为原理的证明，我们使用双标记的15N13C细胞提取物作为IS分析了氨基酸。与最先进的方法相比，对该方法的广泛评估显示出相似的准确性和精确度。我们通过研究激活蛋白激酶R（PKR）样内质网激酶（PERK），即未折叠的蛋白反应的关键组成部分后，哺乳动物细胞中氨基酸代谢的动态响应，证明了该方法的价值。代谢物浓度和同位素特征的整合揭示了PERK活化后氨基酸从头生物合成减少。提议的方法是通用的，可以应用于其他（微生物）生物，