Methodology for the Validation of Isotopic Analyses by Mass Spectrometry in Stable-Isotope Labeling Experiments

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.

稳定同位素标记实验（Stable-isotope labeling experiments, ILEs）被广泛应用于探究代谢网络的拓扑结构与运行机制。在ILEs中采集的同位素数据质量，是确保获得可靠生物学解读的核心前提，但由于缺乏合适的参考物质与相关评价标准，当前的同位素数据评价方法存在明显局限。
本研究提出一套完整的方法学，用于评估代谢系统定量同位素研究所使用的质谱（mass spectrometry, MS）方法。该方法学以标记模式可控的代谢物生物样本为基础，采用多种针对同位素分析的专属质量评价指标，包括同位素异构体（isotopologue）的质量、丰度及质量偏移的准确度与精密度，以及同位素工作范围。
我们将该方法学应用于评估一种全新的氨基酸分析液相色谱-质谱（LC-MS）方法，分别在高分辨质谱仪（工作于全扫描模式的轨道阱（Orbitrap））与低分辨质谱仪（工作于多反应监测（multiple reaction monitoring）模式的三重四极杆质谱仪（triple quadrupole））上完成了测试。
实验结果表明，该方法在宽工作范围内展现出优异的准确度与精密度，同时揭示了基质特异性与仪器模式特异性的分析特征。所提出的方法学能够以简便直接的方式甄别可靠（与不可靠）的同位素数据，并可高效协助排查系统偏差来源，以及识别影响测量整体准确度与精密度的关键因素。
该方法具有普适性，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结果。本研究有望提升同位素测量的可靠性，进而增强ILEs的生物学研究价值。