Broad molecular classification based on stoichiometric ratio relationships has been used extensively to characterize the chemical diversity of aquatic dissolved organic matter (DOM). However, variability in the molecular composition within this classification has remained elusive, thus limiting the interpretation of DOM dynamics, especially with respect to transport versus transformation patterns in response to hydrologic or landscape changes. Here, leveraging high-frequency spatiotemporal sampling during rainfall events at a Critical Zone Observatory project site in Clear Creek, Iowa, we apply a metabolomics-based analysis validated with fragmentation using tandem mass spectrometry to uncover patterns in the molecular features of the DOM composition that were not resolved by classification based on stoichiometric ratios in the chemical formulae. From upstream to downstream sites, beyond the increased aromaticity implied by changes in the stoichiometric ratios, we identified an increased abundance of flavonoids and other phenylpropanoids, two important subgroups of aromatic compounds. The stoichiometric analysis also proposed a localized decline in the abundance of lipid-like compounds, which we attributed specifically to medium-chain and short-chain fatty acids; other lipids such as long-chain fatty acids and sterol lipids remained unchanged. We further determined in-stream molecular transitions and specific compound degradation by capturing changes in the molecular masses of terpenoids, phenylpropanoids, fatty acids, and amino acids. In sum, the metabolomics analysis of the chemical formulae resolved molecular variability imprinted on the stoichiometric DOM composition to implicate key molecular subgroups underlying carbon transport and cycling dynamics in the stream.
