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Antimicrobial resistance is a global threat. The use of biologically active natural products alone or in combination with the clinically proven antimicrobial agents might be a useful strategy to fight the resistance. The scientific hypotheses of this study were twofold: (1) the natural humic substances rich in dicarboxyl, phenolic, heteroaryl, and other fragments might possess inhibitory activity against β-lactamases, and (2) this inhibitory activity might be linked to the molecular composition of the humic ensemble. To test these hypotheses, we used humic substances (HS) from different sources (coal, peat, and soil) and of different fractional compositions (humic acids, hymatomelanic acids, and narrow fractions from solid-phase extraction) for inhibiting serine β-lactamase TEM-1. Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) was used to characterize the molecular composition of all humic materials used in this study. The kinetic assay with chromogenic substrate CENTA was used for assessment of inhibitory activity. The inhibition data have shown that among all humic materials tested, a distinct activity was observed within apolar fractions of hymatomelanic acid isolated from lignite. The decrease in the hydrolysis rate in the presence of most active fractions was 42% (with sulbactam-87%). Of particular importance is that these very fractions caused a synergistic effect (2-fold) for the combinations with sulbactam. Linking the observed inhibition effects to molecular composition revealed the preferential contribution of low-oxidized aromatic and acyclic components such as flavonoid-, lignin, and terpenoid-like molecules. The binding of single low-molecular-weight components to the cryptic allosteric site along with supramolecular interactions of humic aggregates with the protein surface could be considered as a major contributor to the observed inhibition. We believe that fine fractionation of hydrophobic humic materials along with molecular modeling studies on the interaction between humic molecules and β-lactamases might contribute to the development of novel β-lactamase inhibitors of humic nature.

Chronic liver diseases affect more than 1 billion people worldwide and represent one of the main public health issues. Nonalcoholic fatty liver disease (NAFLD) accounts for the majority of mortal cases, while there is no currently approved therapeutics for its treatment. One of the prospective approaches to NAFLD therapy is to use a mixture of natural compounds. They showed effectiveness in alleviating NAFLD-related conditions including steatosis, fibrosis, etc. However, understanding the mechanism of action of such mixtures is important for their rational application. In this work, we propose a new dereplication workflow for deciphering the mechanism of action of the lignin-derived natural compound mixture. The workflow combines the analysis of molecular components with high-resolution mass spectrometry, selective chemical tagging and deuterium labeling, liver tissue penetration examination, assessment of biological activity in vitro, and computational chemistry tools used to generate putative structural candidates. Molecular docking was used to propose the potential mechanism of action of these structures, which was assessed by a proteomic experiment.

We report a novel family of natural lipoglycopeptides produced by Streptomyces sp. INA-Ac-5812. Two major components of the mixture, named gausemycins A and B, were isolated, and their structures were elucidated. The compounds are cyclic peptides with a unique peptide core and several remarkable structural features, including unusual positions of d-amino acids, lack of the Ca2+ -binding Asp-X-Asp-Gly (DXDG) motif, tyrosine glycosylation with arabinose, presence of 2-amino-4-hydroxy-4-phenylbutyric acid (Ahpb) and chlorinated kynurenine (ClKyn), and N-acylation of the ornithine side chain. Gausemycins have pronounced activity against Gram-positive bacteria. Mechanistic studies highlight significant differences compared to known glyco- and lipopeptides. Gausemycins exhibit only slight Ca2+ -dependence of activity and induce no pore formation at low concentrations. Moreover, there is no detectable accumulation of cell wall biosynthesis precursors under treatment with gausemycins.

Direct comparison of high-resolution mass spectrometry (HRMS) data acquired with different instrumentation or parameters remains difficult as the derived lists of molecular species via HRMS, even for the same sample, appear distinct. This inconsistency is the result of inherent inaccuracies caused by instrumental limitations and sample conditions. We propose a method that classifies HRMS data based on the differences in the number of elements between each pair of molecular formulae within the formulae list to preserve the essence of the given sample. The novel metric, Formulae Difference Chains Expected Length (FDCEL), allowed for comparing and classifying samples measured by different instruments. FDCEL metric was implemented for both spectrum quality control and for examination of samples of various nature. We also demonstrate a web application and a prototype for a uniform database for HRMS data serving as a benchmark for future biogeochemical applications.

Interlaboratory comparison on the determination of the molecular composition of humic substances (HS) was undertaken in the framework of IUPAC project 2016-015-2-600. The analysis was conducted using high resolution mass spectrometry, nominally, Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization. Six samples of HS from freshwater, soil, and leonardite were used for this study, including one sample of humic acids (HA) from coal (leonardite), two samples of soil HA (the sod-podzolic soil and chernozem), two samples of soil fulvic acids (FA) (the sod-podzolic soil and chernozem), and one sample of freshwater humic acids (the Suwannee River). The samples were analyzed on five different FTICR MS instruments using the routine conditions applied in each participating laboratory. The results were collected as mass lists, which were further assigned formulae for the determination of molecular composition. The similarity of the obtained data was evaluated using appropriate statistical metrics. The results have shown that direct comparison of discrete stoichiometries assigned to the mass lists obtained by the different laboratories yielded poor results with low values of the Jaccard similarity score – not exceeding 0.56 (not more than 56 % of the similar peaks). The least similarity was observed for the aromatics-rich HA samples from leonardite (coal) and the chernozem soil, which might be connected to difficulties in their ionization. The reliable similarity among the data obtained in this intercomparison study was achieved only by transforming a singular point (stoichiometry) in van Krevelen diagram into a sizeable pixel (a number of closely located stoichiometries), which can be calculated from the population density distribution. The conclusion was made that, so far, these are descriptors of occupation density distribution, which provide the metrics compliant with the data quality requirements, such as the reproducibility of the data measurements on different instruments.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides a unique opportunity for molecular analysis of natural complex mixtures. In many geochemical and environmental studies structure-propertry relations are based solely on the elemental compositional information. Several calculated parameters were proposed to increase reliability of structural attribution, among which aromaticity indices (AI and AImod) are widely used. Herein, we applied a combination of selective labeling reactions in order to obtain direct structural information on the individual components of lignin-derived polyphenolic material. Carboxylic (COOH), carbonyl (C═O), and hydroxyl (OH) groups were enumerated by esterification, reducing, and acetylation reactions, respectively, followed by FTICR MS analyses. Obtained information was enabled to constrain aromaticity accounting for the carbon skeleton only. We found that actual aromaticity of components may be both higher or lower than approximated values depending on the abundance of COOH, C═O, and OH groups. The results are of importance for the geochemical community studying terrestrial NOM with structural gradients.

Accurate and reliable identification of chemical compounds is the ultimate goal of mass spectrometry analyses. Currently, identification of compounds is usually based on the measurement of the accurate mass and fragmentation spectrum, chromatographic elution time, and collisional cross section. Unfortunately, despite the growth of databases of experimentally measured MS/MS spectra (such as MzCloud and Metlin) and developing software for predicting MS/MS fragments in silico from SMILES patterns (such as MetFrag, CFM-ID, and Ms-Finder), the problem of identification is still unsolved. The major issue is that the elution time and fragmentation spectra depend considerably on the equipment used and are not the same for different LC-MS systems. It means that any additional descriptors depending only on the structure of the chemical compound will be of big help for LC-MS/MS-based omics. Our approach is based on the characterization of compounds by the number of labile hydrogen and oxygen atoms in the molecule, which can be measured using hydrogen/deuterium and 16O/18O-exchange approaches. The number of labile atoms (those from -OH, -NH, ═O, and -COOH groups) can be predicted from SMILES patterns and serves as an additional structural descriptor when performing a database search. In addition, distribution of isotope labels among MS/MS fragments can be roughly predicted by software such as MetFrag or CFM-ID. Here, we present an approach utilizing the selection of structural candidates from a database on the basis of the number of functional groups and analysis of isotope labels distribution among fragments. It was found that our approach allows reduction of the search space by a factor of 10 and considerably increases the reliability of the compound identification.

LC-MS is a key technique for the identification of small molecules in complex samples. Accurate mass, retention time, and fragmentation spectra from LC-MS experiments are compared to reference values for pure chemical standards. However, this information is often unavailable or insufficient, leading to an assignment to a list of candidates instead of a single hit; therefore, additional features are desired to filter candidates. One such promising feature is the number of specific functional groups of a molecule that can be counted via derivatization or isotope-exchange techniques. Hydrogen/deuterium exchange (HDX) is the most widespread implementation of isotope exchange for mass spectrometry, while oxygen

In land-based recirculating aquaculture systems (RAS), the accumulation of dissolved organic matter (DOM) can have detrimental effects on water quality impacting the system performance, microbial community, and consequently fish health and welfare. Ozone is used in the RAS water treatment process to improve water quality and remove DOM. However, little is known about the molecular composition of DOM in RAS and its transformation when exposed to ozone. In this study, we performed a detailed molecular characterization of DOM in RAS and explored its transformation induced by ozonation of RAS waters. Ultra-high resolution (UHR) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) was used to characterize the DOM matrix of RAS waters (pump-sump and tanks) and to evaluate its transformation by ozonation. The analysis of DOM extracted from makeup water and feed samples allowed for the determination of DOM sources in RAS prior to ozonation. The CHO and unsaturated group of compounds were the most abundant class found in water samples. On the contrary, the DOM from feed samples was unique and consisted mainly of CHO, CHON and unsaturated group of compounds. After the ozonation of RAS waters, humic-like and unsaturated compounds [positive oxygen subtracted double bond equivalent per carbon (DBE–O)/C)] were decomposed, particularly the CHO-DOM that contained fewer -CH2- features. Fulvic-like compounds and several hundred saturated compounds [negative (DBE–O)/C)] were formed post ozonation, particularly the CHON and CHONS group of compounds that were associated with fish diets, makeup waters and transformation products from the ozonation of the RAS waters. This study showed that the high accuracy of the ultra-high resolution FTICR MS can be applied to characterize and monitor the changes of DOM at a molecular level in RAS waters. To our knowledge, this is the first study where FTICR MS was incorporated for the characterization of DOM and its sources in RAS.

Humification is a ubiquitous natural process of biomass degradation that creates multicomponent systems of nonliving organic matter, including dissolved organic matter (DOM) and humic substances (HS) in water environments, soils, and organic rocks. Despite significant differences in molecular composition, the optical properties of DOM and HS are remarkably similar, and the reason for this remains largely unknown. Here, we employed fluorescence spectroscopy with (sub)picosecond resolution to elucidate the role of electronic interactions within DOM and HS. We revealed an ultrafast decay component with a characteristic decay lifetime of 0.5-1.5 ps and spectral diffusion originating from excitation energy transfer (EET) in the system. The rate of EET was positively correlated to the fraction of aromatic species and tightness of aromatic species packing. Diminishing the number of EET donor-acceptor pairs by reduction with NaBH4 (decrease of the acceptor number), decrease of pH (decrease of the electron-donating ability), or decrease of the average particle size by filtration (less donor-acceptor pairs within a particle) resulted in a lower impact of the ultrafast component on fluorescence decay. Our results uncover the role of electronic coupling among fluorophores in the formation of DOM and HS optical properties and provide a framework for studying photophysical processes in heterogeneous systems of natural fluorophores.