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Introduction: FTICR MS is an exclusive method, which enables exploration of extremely complex natural organic matter (NOM). Routine analysis resolves thousands of CcHhOoNnSs molecular compositions in NOM sample [1]. However, FTICR MS is tolerant to structural isomers. Here we show determination of specific structural sites in individual NOM molecules and isomers in different samples using selective labeling reactions and direct fractionation in combination with FTICR MS. Methods: NOM samples were sealed with NaOD or DCl for skeletal HDX [2]. Deuteromethylation was performed by CD3OD/SOCl2 reaction of NOM. All samples were desalted [3] and fractionated using SPE on ppl cartridges operated at different pH. HDX of mobile protons was performed by incubation in D2O. All samples were analysed using 7T FT MS Bruker Apex Ultra with harmonized cell (Bruker Daltonics). The FTICR MS data were processed using the lab-made “Transhumus” software. Results: Catalytic HDX coupled to FTICR MS was applied to determine structural fragments of individual molecules of NOM and its synthetic analog. Application of HDX showed isomeric lignin-like components, which differ by aromatic ring substitution pattern. Combination of skeletal HDX provides the reliable identification of isomers in various NOM samples isolated even from similar sources. In case of models obtained by oxidative condensation of phenols, the exact structure of components was suggested. Deuteromethylation enabled determination of COOH-groups carried by individual molecules in NOM. This allowed for mapping compounds with close elemental compositions but different protolytic properties on conventional Van Krevelen diagram. The number of COOH-groups matched model structures suggested for major NOM components. Based on this result we performed direct fractionation of NOM on hydrophobic resin performed at different pH. We observe distinct shift of molecular composition from COOH-depleted molecules to polycarboxylic compounds. Conclusions We found that shared molecular compositions identified in different NOM samples correspond to structural isomers. By visualization of labeling results, we discovered relationships between compartments of NOM – oxidation, decarboxylation, condensation, etc, including transformation of CHOS compounds. Due to distinct separation of carboxylic acids according to molecular compositions, the deeper fractionation of hydrophobic components of NOM was performed.