In/postsource fragments (ISFs) arise during electrospray ionization or ion transfer in mass spectrometry when molecular bonds break, generating ions that can complicate data interpretation. Although ISFs have been recognized for decades, their contribution to untargeted metabolomicsparticularly in the context of the so-called "dark matter" (unannotated MS or MS/MS spectra) and the "dark metabolome" (unannotated molecules)remains unsettled. This ongoing debate reflects a central tension: while some caution against overinterpreting unidentified signals lacking biological evidence, others argue that dismissing them too quickly risks overlooking genuine molecular discoveries. These discussions also raise a deeper question: what exactly should be considered part of the metabolome? As metabolomics advances toward large-scale data mining and high-throughput computational analysis, resolving these conceptual and methodological ambiguities has become essential. In this perspective, we propose a refined definition of the "dark metabolome" and present a systematic overview of ISFs and related ion forms, including adducts and multimers. We examine their impact on metabolite annotation, experimental design, statistical analysis, computational workflows, and repository-scale data mining. Finally, we provide practical recommendationsincluding a set of dos and do nots for researchers and reviewersand discuss the broader implications of ISFs for how the field explores unknown molecular space. By embracing a more nuanced understanding of ISFs, metabolomics can achieve greater rigor, reduce misinterpretation, and unlock new opportunities for discovery.

Center for Microbiome Innovation University of California San Diego La Jolla California 92093 United States

CIBER de Diabetes y Enfermedades Metabólicas Asociadas Instituto de Salud Carlos 3 Madrid 28029 Spain

Collaborative Mass Spectrometry Innovation Center Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla California 92093 United States

Department of Biochemistry of Plant Specialized Metabolites Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo Náměstí 542 2 Prague 160 00 Czech Republic

Department of Chemistry Genetics and Medicine and Center for Mass Spectrometry and Metabolic Tracing Washington University St Louis Missouri 63110 United States

Department of Electronic Engineering and IISPV Universitat Rovira i Virgili Tarragona 43007 Spain

Institute for Molecular Systems Biology ETH Zurich Otto Stern Weg 3 Zurich 8093 Switzerland

Lewis Sigler Institute for Integrative Genomics and Department of Chemistry Princeton University Princeton New Jersey 08544 United States

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego 9500 Gilman Drive San Diego California 92093 0751 United States

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