Unsupervised Structural Classification of Dissolved Organic Matter Based on Fragmentation Pathways

Dissolved organic matter (DOM) is considered an essential component of the Earth's ecological and biogeochemical processes. Structural information of DOM components at the molecular level remains one of the most extraordinary analytical challenges. Advances in determination of chemical formulas from the molecular studies of DOM have provided limited indications on structural signatures and potential reaction pathways. In this work, we extend the structural characterization of a wetland DOM sample using precursor and fragment molecular ions obtained by a sequential electrospray ionization-Fourier transform-ion cyclotron resonance tandem mass spectrometry (ESI-FT-ICR CASI-CID MS/MS) approach. The DOM chemical complexity resulted in near 900 precursors (P) and 24 000 fragment (F) molecular ions over a small m/z 261-477 range. The DOM structural content was dissected into families of structurally connected precursors based on neutral mass loss patterns (P + F_1: + C) across the two-dimensional (2D) MS/MS space. This workflow identified over 1900 structural families of DOM compounds based on a precursor and neutral loss (H₂O, CH₄O, and CO₂). The inspection of structural families showed a high degree of isomeric content (numerous identical fragmentation pathways), not discriminable with sole precursor ion analysis. The connectivity map of structural families allows for the visualization of potential biogeochemical processes that DOM undergoes throughout its lifetime. This study illustrates that integrating effective computational tools on a comprehensive high-resolution mass fragmentation strategy further enables the DOM structural characterization.