Definition of Metabolite: Size as a Critical Criterion

Metabolites have traditionally been defined as organic molecules smaller than 1500 daltons (Da). However, recent advances in analytical technologies and chemoinformatics have uncovered a wider chemical diversity, including biologically significant metabolites exceeding this conventional size cutoff─such as polypeptides, glycosphingolipids, and bacterial lipopolysaccharides. Detecting these larger metabolites challenges standard metabolomics approaches and necessitates optimized mass spectrometry acquisition parameters. In this perspective, the analysis of multiple databases (HMDB blood, GNPS, Plant Molecular Network [PMN], Natural Product Atlas [NPA] fungi, NPA bacteria, and MiMeDB) confirms that although most metabolites are below 1000 Da, notable populations exceed this threshold, particularly in bacterial data sets. Furthermore, reanalysis of liquid chromatography-mass spectrometry (LC-MS²) data sets from diverse biological samples, especially bacteria-rich matrices like feces and skin, reveals features (peaks with <i>m</i>/<i>z</i> and retention time) extending beyond 1500 <i>m</i>/<i>z</i>. These findings underscore that metabolites are often larger than commonly recognized in the literature. Therefore, the definition of metabolites should evolve to accommodate their size diversity, ensuring accurate knowledge dissemination to new generations of metabolomics researchers.