Recent improvements in MARS for computer assisted-analysis in untargeted stable isotope labelling studies in metabolomics

Technological advances in mass spectrometry instrumentation have increased the resolution and sensitivity of measurements allowing the detection of less abundant species in matrices. At the same time, the coupling of liquid chromatography with mass spectrometry (LC-MS) has enabled the extra-dimensional separation of compounds, facilitating their detection in untargeted studies. Specifically designed software solutions have become increasing necessary for data analysis when working untargeted, due to the huge amounts of data generated. However, only a limited number of software cover all the steps required in untargeted metabolomics data analysis, also providing tailored computational tools for stable isotope labelling (SIL) analysis. Indeed, complexity increases when SIL studies are performed. Unlabeled and labelled compounds needs to be processed and annotated within the same dataset. In addition, the projection of the identified species in metabolic maps is crucial for the biological interpretation of results. Here, we describe the improvements of the computational workflow for SIL analysis already in place in the previous version of MARS [1] (https://mass-analytica.com/products/mars/) a software solution for untargeted LC-MS untargeted metabolomics analysis. MARS covers the study of uniformly and partially labelled species, considering the most common isotopes used for labelling (e.g. D, 13C, 15N, 34S, 37Cl). The annotation of labelled metabolites can be performed at MS and MS/MS level. Indeed, the MS/MS spectra of labelled metabolites are generated automatically from those of native species and stored in a database for identification purpose. In addition, optimization of the isotopic pattern clustering based on instrument resolution was recently added. Native and labelled compounds identified as different ionization adducts and at different level (MS or MS/MS) can be inspected together or separately for an easier investigation and for projection into the metabolic maps included in the package. Ready-to-use databases derived by commonly used tracers are available for download. In conclusion, we implemented user-friendly informatic tools in MARS to support and facilitate the data analysis in stable isotope labelling studies in metabolomics. The entire workflow and algorithms are elucidated using an appropriate showcase.

[1] Goracci L., Tiberi P., Di Bona S., Bonciarelli S., Passeri G. I., Piroddi M., Moretti S., Volpi C., Zamora I., Cruciani G.; MARS: a multi-purpose software for untargeted LC-MS-based metabolomics and exposomics. Anal. Chem., 2024, 96, 4, 1468–1477. https://doi.org/10.1021/acs.analchem.3c03620