In/post-source 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 don’ts 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.

Abstract. Metabolite and small molecule identification via tandem mass spectrometry (MS/MS) involves matching experimental spectra with prerecorded spectra

The deposition of matrix compounds significantly influences the effectiveness of matrix-assisted laser desorption/ionization (MALDI) Mass Spectrometry Imaging (MSI) experiments, impacting sensitivity, spatial resolution, and reproducibility. Dry deposition methods offer advantages by producing homogeneous matrix layers and minimizing analyte delocalization without the use of solvents. However, refining these techniques to precisely control matrix thickness, minimize heating temperatures, and ensure high-purity matrix layers is crucial for optimizing MALDI-MSI performance. Here, we present a novel approach utilizing low-temperature thermal evaporation (LTE) for organic matrix deposition under reduced vacuum pressure. Our method allows for reproducible control of matrix layer thickness, as demonstrated by linear calibration for two organic matrices, 2,5-dihydroxybenzoic acid (DHB) and 1,5-diaminonaphthalene (DAN). The environmental scanning electron microscopy images reveal a uniform distribution of small-sized matrix crystals, consistently on the sub-micrometer scale, across tissue slides following LTE deposition. Remarkably, LTE serves as an additional purification step for organic matrices, producing very pure layers irrespective of initial matrix purity. Furthermore, stability assessment of MALDI-MSI data from mouse brain sections coated with LTE-deposited DHB or DAN matrix indicates minimal impact on ionization efficiency, signal intensity, and image quality even after storage at −80 °C for 2 weeks, underscoring the robustness of LTE-deposited matrices for MSI applications. Comparative analysis with the spray-coating method highlights several advantages of LTE deposition, including enhanced ionization, reduced analyte diffusion, and improved MSI image quality.