Leveraging Dissolved Organic Matter Collections as a Natural Chemical Library to Link Molecular Traits with Cellular Morphological Responses

Dissolved organic matter (DOM) exhibits a highly complex molecular composition and maintains ecosystem stability, acting as a crucial interface between biotic and abiotic processes. Although DOM's molecular complexity and biological effects are widely studied, most investigations use targeted bioassays, examining specific responses and linking molecular features only to predefined biological outcomes when assessing potential bioactive components. Here, we analyzed International Humic Substances Society (IHSS) reference standards of natural organic matter (NOM) and humic fractions, including humic acids (HAs) and fulvic acids (FAs), using ultrahigh resolution Fourier- transform ion cyclotron resonance mass spectrometry (FT-ICR MS) alongside the Cell Painting (CP) assay, a multiplexed, image-based morphological profiling method. The chemical composition of IHSS samples was influenced by fractionation methods and environmental sources. HAs exhibited stronger aliphatic and aromatic characteristics, whereas FAs and NOM extracted by reverse osmosis were more oxidized. Distinct molecular patterns were observed among terrestrial HAs, Pony Lake FAs, terrestrial FAs, and other fractions. In the CP assay, the most hydrophobic humic substances induced the most pronounced morphological changes. Linking chemical features with morphological outcomes suggested lipid-like compounds and nitrogen-rich aromatic species as likely contributors. This integrative approach provides preliminary molecular leads for further isolation, structural characterization, and mechanistic studies of DOM bioactivity.