5 – Metabolite Identification Confidence Levels for Global Untargeted Molecular Omics

Known and Unknown Metabolites

Given that metabolites lack a genetic template such as that for proteins, metabolomics databases are currently considered incomplete. As such, we observe both known and unknown features in global, untargeted metabolomics studies.

In an effort communicate the confidence of annotation and identification results for known features in global, untargeted metabolomics studies, we use an evidence-based classification system (shown below) that is founded on metabolite identification discussions in the metabolomics community.

MS Validation Levels of Detail

Level Descriptions

We search multiple experimental and in-silico metabolite databases to generate candidate identifications. Inherently, isomeric features may match several candidates. As a result, we review our extensive in-house library to reduce the number of candidates and/or increase the confidence of candidates when possible. While these databases and libraries can guide annotations, it may not always be possible to reduce the results of an observed feature (metabolite) to a single identification candidate. For such metabolites, multiple identification candidates will be provided in our reports.

Level 5 – Unique FeatureOnce a prioritized list of peaks from global, untargeted analysis (incl. via data alignment and analysis multivariate statistical analyses, self-organizing maps, pairwise comparisons, ANOVA, etc.) has been established, unique features are identified via exact mass measurement accuracy. (Unique mass-to-charge ratio and retention time)
Level 4 – Molecular FormulaMolecular formula identification of features is completed via isotope abundance distribution, charge state and adduct ion determination.
Level 3 – Tentative StructureTentative structural identification includes a unique match of the parent ion (MS1) data searched through literature and/or libraries and databases.
Level 2 – Putative IdentificationPutative identification reveals probable structure using fragmentation data from literature and/or libraries and databases.
Level 1 – Validated IdentificationIdentification is fully validated by confirming the structure and metabolite assignment using a reference standard.

References:

  1. Schrimpe-Rutledge, A.C., Codreanu, S.G., Sherrod, S.D. et al. J. Am. Soc. Mass Spectrom. (2016) 27: 1897. https://doi.org/10.1007/s13361-016-1469-y
  2. May, J.C., McLean, J.A. Annu. Rev. Anal. Chem. 2016. 9:387–409. https://doi.org/10.1146/annurev-anchem-071015-041734