Identification of unknown metabolites constitutes a major bottleneck in the metabolomics workflow. Even though the use of spectral databases to match the experimental MS/MS spectra of the unknown metabolites is the most common approach for their characterization, the number of metabolites and other compounds with MS/MS spectra in these databases is limited. Therefore, other tools that rely on the spectral similarity of molecules containing similar functional groups have become very popular to gain insight about the chemical structure of unknown metabolites. In this presentation, the use of the natural and artificially generated stable isotopes of molecules are proposed as complementary methods for the identification of unknowns. The isotopic distribution and the mass defect of a metabolic feature are intrinsic characteristics of the atoms that can provide useful information about the chemical composition of an unknown or the mechanistic analysis of its fragmentation spectra. Moreover, the detailed analysis of the isotopic distribution of a metabolic feature may make emerge multiple charged ions. For the generation of a valuable resource for the identification of unknown metabolites, uniformly-labeled microorganisms extracts were analyzed together with their unlabeled analogues in an untargeted way. This approach, apart of generate a vast number of MS/MS data of uniformly-labeled metabolites for their use in targeted metabolomics, produced hundreds of pairs of unlabeled versus labeled MS/MS spectra of metabolites that can be used for the identification of unknows. The comparison of the m/z shifts between the unlabeled and the labeled metabolite allows for the determination of the total number of carbon atoms of the parent ion and all the fragments. The suitability of this apporach for the identification of unknowns is finally demonstrated by the characterization of 3 unknowns whose MS/MS was not present in any spectral database. Since the generation of spectral databases requires a high economic and infrastructural effort, the use of different isotope-labeled microorganisms arises as a valuable complementary resource for the identification of unknown molecules in metabolomics.

Identifying metabolites using mass spectrometry and stable isotopes

2019 Fall National Meeting

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