ISotopologue mixtures for Quantitative Mass Spectrometry: Application to metabolomics – IS-QMS

Metabolomics seeks to define the complete composition of small molecular weight metabolites in biological samples. It offers enormous potential for the identification of biomarkers to follow the evolution of an individual, to detect a pathology as early as possible and to predict and follow the response to treatment. Today, untargeted metabolomics based on high-resolution mass spectrometry coupled to liquid chromatography (LC-HRMS) remains semi-quantitative with results generally expressed as intensities or peak areas for hundreds or even thousands of signals. Unfortunately, the large number of co-eluting species in complex biological extracts may affect the reliability of this approach.
To access more robust and ideally interoperable data, quantitative MS measurements most often involve the use of stable isotope labeled internal standard (SIL-IS) added to each sample in association with at least one calibration curve. A typical workflow includes sample injections for the cohort itself, along with injections for standard curves and quality controls that can account for up to 20% of total analytical time, resulting in a loss of time and money. In addition, the practice of such a quantitative approach is restricted to the analysis of a few dozen metabolites. This is partly due to the cost of commercially available SIL-IS but also to the cumbersome nature of quantitative measurements, requiring the setting up of external ranges with a minimum of 6 points for each metabolite to be quantified.
To allow the quantitative characterization of complex biological samples, with the production of robust and interoperable data for an extended metabolome coverage, the IS-QMS project proposes to develop an internal multi-point calibration approach based on the use of different stable isotope labeled-versions (isotopologues) of each metabolite to quantify. The innovative analytical strategy is based on the use of at least three isotopologues per metabolite, each isotopologue being used as a calibrator, to generate direct in-sample calibration curves.
To reach this objective, we propose to develop original and labeled chemical tools: (1) labeled-metabolites for quantification within global approaches and (2) labeled-tags for the quantitative exploration of sub-metabolomes.
Based on our preliminary results, our goal is to produce a library of isotopologue mixtures (IsoMixes), using a simple, one-step, metal-catalyzed hydrogen/deuterium exchange (HDX) reaction, performed on the target metabolite, which allows a stable incorporation of deuterium on carbon atoms. It has been shown that mixtures of isotopologues of the same metabolite can be obtained by modulating the reaction conditions applied which, when added to a sample, will give in-situ calibration curves. The same labeling strategy will be applied to incorporate deuterium atoms in the chemical structure of optimized tagging-agents: Tag-IsoMixes.
Once produced, these isotopic tools (IsoMixes and Tag-IsoMixes) will be used for quantitative measurements of a large panel of metabolites in biological samples. The performances of the novel approach will be compared to the gold standard methods currently used in MS-based metabolomics studies in our laboratories.
This novel concept breaks with the current state of the art and the expected advances are multiple: - Limiting the impact of analytical deviations, - Improving the robustness of the quantification, - Obtaining quantitative data on not yet identified metabolites, - Shortening the time needed to analyze large number of samples, - and contributing to making data more easily exchangeable and reusable in compliance with the FAIR data strategy.
This project follows the strategic plan of MetaboHUB (MTH), whose goal is to analyze large cohorts of medical samples in a harmonized and interoperable manner to fulfill FAIR principles.