Methodology for the Validation of Isotopic Analyses by Mass Spectrometry in Stable-Isotope Labeling Experiments
Methodology for the Validation of Isotopic Analyses by Mass Spectrometry in Stable-Isotope Labeling Experiments
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DOI：
10.1021/acs.analchem.7b03886
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发表时间：
2018-02-06
影响因子：
7.4
通讯作者：
Portais, Jean-Charles
中科院分区：
文献类型：
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作者：
Heuillet, Maud;Bellvert, Floriant;Portais, Jean-Charles
稳定同位素标记实验（ILEs）被广泛用于研究代谢网络的拓扑结构和运作。在ILE中收集的同位素数据的质量对于确保可靠的生物学解释至关重要，但由于缺乏合适的参考材料和相关的评估标准，目前的评估方法受到限制。在这项工作中，我们提出了一个完整的方法来评估质谱（MS）方法用于定量同位素研究的代谢系统。该方法基于含有具有受控标记模式的代谢物的生物样品，利用特定于同位素分析的不同质量指标（同位素体质量、丰度和质量偏移以及同位素工作范围的准确度和精密度）。我们应用这种方法来评估一种新的LC-MS方法分析氨基酸，这是测试高分辨率（Orbitrap操作在全扫描模式）和低分辨率（三重四极杆操作在多反应监测模式）质谱仪。结果表明，在很大的工作范围内，具有出色的准确度和精密度，并显示出特定于基质和特定于模式的特性。所提出的方法可以识别可靠的（和不可靠的）同位素数据在一个简单和直接的方式，并有效地支持系统偏差的来源，以及影响测量的整体准确度和精度的主要因素的识别。这种方法是通用的，可用于验证不同基质、分析平台、标记元素或代谢物类别的同位素分析。预计这将加强同位素测量的可靠性，从而提高ILE的生物学价值。
Stable-isotope labeling experiments (ILEs) are widely used to investigate the topology and operation of metabolic networks. The quality of isotopic data collected in ILEs is of utmost importance to ensure reliable biological interpretations, but current evaluation approaches are limited due to a lack of suitable reference material and relevant evaluation criteria. In this work, we present a complete methodology to evaluate mass spectrometry (MS) methods used for quantitative isotopic studies of metabolic systems. This methodology, based on a biological sample containing metabolites with controlled labeling patterns, exploits different quality metrics specific to isotopic analyses (accuracy and precision of isotopologue masses, abundances, and mass shifts and isotopic working range). We applied this methodology to evaluate a novel LC-MS method for the analysis of amino acids, which was tested on high resolution (Orbitrap operating in full scan mode) and low resolution (triple quadrupole operating in multiple reaction monitoring mode) mass spectrometers. Results show excellent accuracy and precision over a large working range and revealed matrix-specific as well as mode-specific characteristics. The proposed methodology can identify reliable (and unreliable) isotopic data in an easy and straightforward way and efficiently supports the identification of sources of systematic biases as well as of the main factors that influence the overall accuracy and precision of measurements. This approach is generic and can be used to validate isotopic analyses on different matrices, analytical platforms, labeled elements, or classes of metabolites. It is expected to strengthen the reliability of isotopic measurements and thereby the biological value of ILEs.