Unsupervised Structural Classification of Dissolved Organic Matter Based on Fragmentation Pathways

Abstract

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_n-1 + F_1:n + 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.

Keywords: DOM; ESI-FT-ICR MS/MS; core fragment; network; neutral loss; precursor.

Figure 1.

ESI-FT-ICR MS broadband spectrum of the SPE-DOM sample and expanded view of the m/z range 406–410 shown in the inset (A). Van Krevelen plot obtained after the chemical formula assignment of mass signals with black arrows describing DOM reaction pathways previously suggested by Kim et al. CHO, CHOS, CHON, and CHONS compound classes are represented in green, orange, blue, and gray colors, respectively (B). The section of an MS/MS spectrum showing [M – H]⁻ precursor ions isolated at nominal mass 313. Assigned molecular formulas are displayed with heteroatoms indicated with the color code (C). Typical MS/MS spectrum of the precursors isolated at nominal m/z 313 with annotated common neutral losses observed in DOM (D). Note that single peaks showed at nominal masses may comprise an envelope of multiple mass signals. For instance, nine peaks resulting from the CO₂ loss of precursors fragmented at m/z 313 are shown at m/z 269 (Panel D, inset).

Figure 2.

2D MSMS plots generated after the chemical formula assignment of ion signals obtained from the FT-ICR CASI-CID MS/MS experiments.

Figure 3.

Conceptual models designed to compute ordered fragmentation pathways (panel A) and find structural families in DOM based on sequential matching of fragmentation pathways (Panel B). Note that for the precursor P1 to be considered in a family, its ion mass should match (1 mDa tolerance) the mass of the first fragment in P₂’s fragmentation pathway.

Figure 4.

Number of covered precursors, core and intermediate fragments by the model P_n−1 + F_1:n + C (A), distribution of the number of families per family size (B), and families per oxygen class of the uppermost precursor (compound with the highest oxygen content in the family) (C), respectively, for the CHO class.

Figure 5.

2D MS/MS visualization of a characteristic DOM family of six precursors (Panel A). Chemical unity (H₂O, CH₄O, and CO₂) differences among precursors are shown using a color code. Fragmentation pathways described as neutral losses are also shown as colored bars. Van Krevelen plot (B) of CHO class compounds obtained from the MS₁ experiment highlighting the compositional nature of the structural family.

Figure 6.

View of the three main clusters observed in the network of neutral-loss-based structurally connected DOM precursors for the CHO class. Precursor molecules are described by nodes and the family indexes are shown as edges. An expanded view of 14 interconnected DOM families is shown in the inset. A comprehensive web-based network can be found at https://github.com/Usman095/Graph-DOM.