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Cyanobacteria are promising microbial hosts for production of various industrially relevant compounds, such as succinate, a central metabolite of the tricarboxylic acid cycle (TCA). Cyanobacteria have been engineered to produce succinate during photoautotrophic growth, and are also able to secrete it during anoxic fermentation conditions. It has been assumed that under anoxic darkness, succinate can be formed by reduction of fumarate catalyzed by the succinate dehydrogenase complex (SDH), however, no characterization of SDH regarding this activity has been performed. In this study, we address this issue by generating strains of the unicellular cyanobacteriumSynechocystisPCC 6803 (Synechocystis) deficient in one or several subunits of SDH, and investigating succinate accumulation in these strains during dark anaerobic fermentation. The results showed higher succinate accumulation in SDH deletion strains than in the wild type, indicating a succinate dehydrogenase activity of SDH rather than fumarate reduction under these conditions. We further explored the possibility of another potential route for succinate formation from fumarate via L-aspartate oxidase (Laspo). The gene encoding Laspo inSynechocystiscould not be inactivated, indicating an essential function for this enzyme. Using purifiedSynLaspo, we could demonstratein vitrothat in addition to L-aspartate oxidation the enzyme exhibits an L-aspartate-fumarate oxidoreductase activity. We therefore suggest that reduction of fumarate to succinate during anoxic darkness can be a byproduct of the Laspo reaction, which is the first step in biosynthesis of NAD cofactors. This work contributes to the understanding of cyanobacterial TCA cycle for future engineering and sustainable production of dicarboxylic acids.

This is a peer-reviewed accepted manuscript version of an article that was published as: Duncan, K.D., Hawkes, J.A., Berg, M., Clarijs, B., Gill, C.G., Bergquist, J., Lanekoff, I., & Krogh, E.T. (2022). Membrane sampling separates naphthenic acids from biogenic dissolved organic matter for direct analysis by mass spectrometry. Environmental Science & Technology, 56(5), 3096-3105. https://doi.org/10.1021/acs.est.1c07359 Oil sands process waters can release toxic naphthenic acids (NAs) into aquatic environments. Analytical techniques for NAs are challenged by sample complexity and interference from naturally occurring dissolved organic matter (DOM). Herein, we report the use of a poly(dimethylsiloxane) (PDMS) polymer membrane for the on-line separation of NAs from DOM and use direct infusion electrospray ionization mass spectrometry to yield meaningful qualitative and quantitative information with minimal sample cleanup. We compare the composition of membrane-permeable species from natural waters fortified with a commercial NA mixture to those derived from weak anion exchange solid-phase extraction (SPE) using high-resolution mass spectrometry. The results show that SPE retains a wide range of carboxylic acids, including biogenic DOM, while permeation through PDMS was selective for petrogenic classically defined NAs (CnH2n+zO2). A series of model compounds (log Kow ∼1–7) were used to characterize the perm-selectivity and reveal the separation is based on hydrophobicity. This convenient sample cleanup method is selective for the O2 class of NAs and can be used prior to conventional analysis or as an on-line analytical strategy when coupled directly to mass spectrometry. Research was supported through the Natural Sciences and Engineering Research Council of Canada (NSERC Discovery Grant 2016-06454 ETK) and MITACS Career Connect Internship IT10266 (MB). The work at UU was supported by an Olsson Borgh Scholarship (Grant 130305807) (JAH), the Swedish Research Council (VR; 2018-04618, JAH) and the Swedish Foundation for Strategic Research (ICA-6 and ITM, IL). Post-print version Includes supporting information. https://viurrspace.ca/bitstream/handle/10613/26898/GillKrogh2022EST.pdf?sequence=4

Untargeted molecular analyses of complex mixtures are relevant for many fields of research, including geochemistry, pharmacology, and medicine. Ultrahigh-resolution mass spectrometry is one of the most powerful tools in this context. The availability of open scripts and online tools for specific data processing steps such as noise removal or molecular formula assignment is growing, but an integrative tool where all crucial steps are reproducibly evaluated and documented is lacking. We developed a novel, server-based tool (ICBM-OCEAN

High-resolution mass spectrometry (HRMS) elucidates the molecular composition of dissolved organic matter (DOM) through the unequivocal assignment of molecular formulas. When HRMS is used as a detector coupled to high performance liquid chromatography (HPLC), the molecular fingerprints of DOM are further augmented. However, the identification of eluting compounds remains impossible when DOM chromatograms consist of unresolved humps. Here, we utilized the concept of mathematical chromatography to achieve information reduction and feature extraction. Parallel Factor Analysis (PARAFAC) was applied to a dataset describing the reverse-phase separation of DOM in headwater streams located in southeast Sweden. A dataset consisting of 1355 molecular formulas and 7178 mass spectra was reduced to five components that described 96.89% of the data. Each component summarized the distinct chromatographic elution of molecular formulas with different polarity. Component scores represented the abundance of the identified HPLC features in each sample. Using this chemometric approach allowed the identification of common patterns in HPLC-HRMS datasets by reducing thousands of mass spectra to only a few statistical components. Unlike in principal component analysis (PCA), components closely followed the analytical principles of HPLC-HRMS and therefore represented more realistic pools of DOM. This approach provides a wealth of new opportunities for unravelling the composition of complex mixtures in natural and engineered systems.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehy-droprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Sea spray aerosol is the largest natural source of aerosol to Earth's atmosphere with significant impacts on climate. Despite this, estimates of the impact of sea spray aerosol on Earth's radiation budget are highly uncertain due to an overall lack of understanding of the physical and chemical factors controlling its composition. Critically, results from studies probing the importance of oceanic biological activity on the amount and type of organic matter present in nascent sea spray aerosol have been ambiguous. Some field studies have shown a relationship between the organic fraction of sea spray aerosol and oceanic primary productivity while others have reported no such relationships. Given this, we have probed the composition of seawater and nascent sea spray aerosol during a phytoplankton bloom in the North Atlantic using a novel liquid chromatography-mass spectrometry method. We observed that the composition of dissolved organic matter present in seawater changed as the phytoplankton bloom progressed over an 18 day period. Further, we observed changes to both the chemical composition of the organic matter present in seawater and the chemical composition of the organic matter present in the sea spray aerosol despite the organic matter mass fraction of the aerosol remaining unchanged. More specifically, we observed that the nascent sea spray aerosol became progressively more enriched in surface-active organic substances as the bloom progressed and that the sea spray aerosol had a distinct organic matter composition compared to the seawater. Thus, our work provides additional insight into the biological dependence of nascent sea spray aerosol composition.

Despite our growing understanding of the global carbon cycle, scientific consensus on the drivers and mechanisms that control dissolved organic carbon (DOC) turnover in aquatic systems is lacking, hampered by the mismatch between research that approaches DOC reactivity from either intrinsic (inherent chemical properties) or extrinsic (environmental context) perspectives. Here we propose a conceptual view of DOC reactivity in which the combination of intrinsic and extrinsic factors controls turnover rates and determines which reactions will occur. We review three major types of reactions (biological, photochemical, and flocculation) from an intrinsic chemical perspective and further define the environmental features that modulate the expression of chemically inherent reactivity potential. Finally, we propose hypotheses of how extrinsic and intrinsic factors together shape patterns in DOC turnover across the land-to-ocean continuum, underscoring that there is no intrinsic DOC reactivity without environmental context. By acknowledging the intrinsic–extrinsic control duality, our framework intends to foster improved modeling of DOC reactivity and its impact on ecosystem services.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids (1 and 2) and nine known compounds (3–11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1–4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Thermokarst activity at permafrost sites releases considerable amounts of ancient carbon to the atmosphere. A large part of this carbon is released via thermokarst ponds, and fungi could be an important organismal group enabling its recycling. However, our knowledge about aquatic fungi in thermokarstic systems is extremely limited. In this study, we collected samples from five permafrost sites distributed across circumpolar Arctic and representing different stages of permafrost integrity. Surface water samples were taken from the ponds and, additionally, for most of the ponds also the detritus and sediment samples were taken. All the samples were extracted for total DNA, which was then amplified for the fungal ITS2 region of the ribosomal genes. These amplicons were sequenced using PacBio technology. Water samples were also collected to analyze the chemical conditions in the ponds, including nutrient status and the quality and quantity of dissolved organic carbon. This dataset gives a unique overview of the impact of the thawing permafrost on fungal communities and their potential role on carbon recycling., Measurement(s)carbon dioxide • methane • dissolved organic carbon • iron(2+) • iron(3+) • total phosphorous • Nitrate Measurement • sulfate • nitrite • ammonium • dissolved nitrogen atom in water • specific ultraviolet absorbance SUVA254 • spectral slope S289 • freshness index BIX • humification index HIX • fluorescence index FI • hydrogen to carbon ratio • oxygen to carbon ratio • m/zTechnology Type(s)gas chromatography • total organic carbon analyzer • spectrophotometer • ion chromatography • total nitrogen analyzer • spectrofluorimeter • high-resolution mass spectrometryFactor Type(s)sampling siteSample Characteristic - Organismfungal sp.Sample Characteristic - Environmentpermafrost • pond water • sediment surfaceSample Characteristic - LocationSiberia • Sweden • Region of Northern Quebec • State of Alaska • Greenland Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.14896713

Thermokarst activity at permafrost sites releases considerable amount of ancient carbon to the atmosphere. A large part of this carbon is released via thermokarst ponds, and fungi could be an important organismal group enabling its recycling. However, our knowledge about aquatic fungi growing in thermokarstic systems is extremely limited. In this study, we collected samples from five permafrost sites distributed across circumpolar Arctic and representing a gradient of permafrost integrity. Samples were taken from the ponds surface water, the detritus and the sediment at the bottom of the ponds. These samples were extracted for total DNA, which was then amplified using primers targeting the fungal ITS2 region of the ribosomal genes. These amplicons were sequenced using PacBio technology. Surface water samples were also collected to analyze the chemical conditions in the ponds, including nutrient status and the quality and quantity of dissolved organic carbon. This dataset gives a unique overview of the impact of the thawing permafrost on fungal communities and their potential role on carbon recycling.

N-nitrosodimethylamine (NDMA) is a disinfection byproduct that has been classified as probable human carcinogen by the US Environmental Protection Agency. According to the published literature, natural dissolved organic matter (DOM) can be a source of NDMA precursors in drinking water. New advances in chemical characterization of DOM with high resolution mass spectrometry (HRMS) are allowing researchers to understand these ultra-complex mixtures. The objective of this study is to investigate analytical methodologies based on HRMS to explore NDMA formation from natural waters. To this aim, different waters from drinking water reservoirs in Spain containing NDMA precursors (quantified by means of NDMA formation potential) in concentrations between 17 and 60 ng/L have been studied. The workflow includes DOM solid-phase extraction and Orbitrap analysis with and without chromatographic separation. Here, we show that the molecular composition of DOM across the studied drinking water reservoirs is correlated with the NDMA formation potential. In particular, we found that NDMA formation potential is associated with compounds with high hydrogen saturation (H/C ≥ 1.5), corresponding also to reservoirs with higher background nutrient concentrations and wastewater indicators. Further chromatographic fractionation did not allow better definition of these possible precursors as they were present in different fractions of the chromatogram, suggesting that they were isomerically complex.

Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

In this paper we investigate interferences that appear in molecular mass spectra from aquatic samples. The interferences are identified as doubly charged ions originating from high molecular weight material, which is especially abundant in terrestrial samples. The interferences could be incorrectly assigned to singly charged formulas with high aromaticity and heteroatom content, as the mass error from such formulas can be less than 1 ppm. We propose a strategy for filtering the interference peaks from mass lists based on the presence of their equivalent isotopologue peaks at mass defects of similar to 0.5 Da.

Inland waters receive and process large amounts of colored organic matter from the terrestrial surroundings. These inputs dramatically affect the chemical, physical, and biological properties of water bodies, as well as their roles as global carbon sinks and sources. However, manipulative studies, especially at ecosystem scale, require large amounts of dissolved organic matter with optical and chemical properties resembling indigenous organic matter. Here, we compared the impacts of two leonardite products (HuminFeed and SuperHume) and a freshly derived reverse osmosis concentrate of organic matter in a set of comprehensive mesocosm‐ and laboratory‐scale experiments and analyses. The chemical properties of the reverse osmosis concentrate and the leonardite products were very different, with leonardite products being low and the reverse osmosis concentrate being high in carboxylic functional groups. Light had a strong impact on the properties of leonardite products, including loss of color and increased particle formation. HuminFeed presented a substantial impact on microbial communities under light conditions, where bacterial production was stimulated and community composition modified, while in dark potential inhibition of bacterial processes was detected. While none of the browning agents inhibited the growth of the tested phytoplankton Gonyostomum semen, HuminFeed had detrimental effects on zooplankton abundance and Daphnia reproduction. We conclude that the effects of browning agents extracted from leonardite, particularly HuminFeed, are in sharp contrast to those originating from terrestrially derived dissolved organic matter. Hence, they should be used with great caution in experimental studies on the consequences of terrestrial carbon for aquatic systems., Marie Curie International Outgoing Fellowship, Swedish Research Council Formas http://dx.doi.org/10.13039/501100001862, Knut and Alice Wallenberg Foundation http://dx.doi.org/10.13039/501100004063

Rust fungi cause epidemics that threaten the production of important plant species, such as wheat and soy. Melampsora larici-populina (Mlp) causes the poplar rust and encodes at least 1184 candidate effectors (CEs) whose functions are poorly known. In this study, we sequenced the transcriptome and used mass spectrometry to analyze the metabolome of Arabidopsis plants constitutively expressing 14 Mlp CEs and of a control line to discover alterations leading to plant susceptibility. We found 2299 deregulated genes across the experiment. Genes involved in pattern-triggered immunity, such as FRK1, PR1, RBOHD, and WRKY33, as well as AUX/IAA genes were down-regulated. We further observed that 680 metabolites were deregulated in at least one CE-expressing transgenic line, with “highly unsaturated and phenolic compounds” and “peptides” enriched among down- and up-regulated metabolites. Interestingly, transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Thus, our results uncouple effector sequence similarity and function. This supports that effector functional investigation in the context of their virulence activity and effect on plant susceptibility requires the investigation of the individual effector and precludes generalization based on sequence similarity.

Autotrophic dissolved organic matter (DOM) is central to the carbon biogeochemistry of aquatic systems, and the full complexity of autotrophic DOM has not been extensively studied, particularly by high-resolution mass spectrometry (HRMS). Terrestrial DOM tends to dominate HRMS studies in freshwaters due to the propensity of such compounds to ionize by negative mode electrospray, and possibly also because ionizable DOM produced by autotrophy is decreased to low steady-state concentrations by heterotrophic bacteria. In this study, we investigated the character of DOM produced by the widespread cyanobacteria Microcystis aeruginosa using high-pressure liquid chromatography—electrospray ionization—high-resolution mass spectrometry. M. aeruginosa produced thousands of detectable compounds in axenic culture. These compounds were chromatographically resolved and the majority were assigned to aliphatic formulas with a broad polarity range. We found that the DOM produced by M. aeruginosa was highly susceptible to removal by heterotrophic freshwater bacteria, supporting the hypothesis that this autotroph-derived organic material is highly labile and accordingly only seen at low concentrations in natural settings.

Rust fungi are plant pathogens that cause epidemics that threaten the production of important plant species, such as wheat, soy, coffee and poplar.Melampsora larici-populina(Mlp) causes the poplar rust and encodes at least 1 184 candidate effectors (CEs), however their functions are poorly known. In this study, we usedArabidopsisplants constitutively expressing CEs ofMlpto discover processes targeted by these fungal proteins. For this purpose, we sequenced the transcriptome and used mass spectrometry to analyse the metabolome ofArabidopsisplants expressing individually one of the 14 selected CEs and of a control line. We found 2 299 deregulated genes across the experiment. Among the down-regulated genes, the KEGG pathways “MAPK signaling pathway” and “Plant-pathogen interaction” were respectively over-represented in six and five of the 14 transgenic lines. Moreover, genes related to hormone response and defense were down-regulated across all transgenic lines are. We further observed that there were 680 metabolites deregulated in at least one CE-expressing transgenic line, with highly unsaturated and phenolic compounds enriched in up-regulated metabolites and peptides enriched among down-regulated metabolites. Interestingly, we found that transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Taken together, our results indicate that the sequence of effectors and their belonging to families may not be a good predictor of their impact on the plant.ImportanceRust fungi are plant pathogens that threaten the production of important crops, including wheat, soy, coffee and poplar. Effectors are used by pathogens to control the host, however in the case ofMelampsora larici-populina, the causal agent of the poplar rust, and other rust fungi these proteins are poorly known. We usedArabidopsisplants expressing candidate effectors (CEs) ofMlpto better understand the interaction between this pathogen and its hosts. We found that expression of unrelated CEs led to similar patterns of gene and metabolite deregulation, while transgenic lines expressing CEs belonging to the same family showed different groups of different genes and metabolites deregulated. Thus, our results suggest that functional annotation of effectors based on sequence similarity may be misleading.

Dissolved organic matter (DOM) composition exerts a direct control on its degradation and subsequent persistence in aquatic ecosystems. Yet, under certain conditions, the degradation patterns of DOM cannot be solely explained by its composition, highlighting the relevance of environmental conditions for DOM degradation. Here, we experimentally assessed the relative influence of composition vs. environment on DOM degradation by performing degradation bioassays using three contrasting DOM sources inoculated with a standardized bacterial inoculum under five distinct environments. The DOM degradation kinetics modeled using reactivity continuum models showed that composition was more important than environment in determining the bulk DOM decay patterns. Changes in DOM composition resulted from the interaction between DOM source and environment. The role of environment was stronger on shaping the bacterial community composition, but the intrinsic nature of the DOM source exerted stronger control on the DOM degradation function Research Funding: Agència de Gestió d'Ajuts Universitaris i de Recerca. Grant Number: BP2016‐00215 European Commission. Grant Number: Marie Sklodowska‐Curie 839709 Ministerio de Ciencia e Innovación. Grant Number: FJC2018‐037791‐I Spanish Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). Grant Number: FEDER‐MCIU‐AEI/CGL2017‐86788‐C3 Spanish Ministry of Economy and Competitiveness. Grant Number: CGL2014‐61771‐EXP

Electrospray ionization (ESI) operating in the negative mode coupled to high-resolution mass spectrometry is the most popular technique for the characterization of dissolved organic matter (DOM). The vast molecular heterogeneity and the functional group diversity of this complex mixture prevents the efficient ionization of the organic material by a single ionization source, so the presence of uncharacterized material is unavoidable. The extent of this poorly ionizable pool of carbon is unknown, is presumably variable between samples, and can only be assessed by the combination of analysis with a uniform detection method. Charged aerosol detection (CAD), whose response is proportional to the amount of nonvolatile material and is independent from the physicochemical properties of the analytes, is a suitable candidate. In this study, a fulvic acid mixture was fractionated and analyzed by high-pressure liquid chromatography–mass spectrometry in order to investigate the polarity and size distributions of highly and poorly ionizable material in the sample. Additionally, DOM samples of terrestrial and marine origins were analyzed to evaluate the variability of these pools across the land–sea aquatic continuum. The relative response factor values indicated that highly ionizable components of aquatic DOM mixtures are more hydrophilic and have lower molecular weight than poorly ionizable components. Additionally, a discrepancy between the samples of terrestrial and marine origins was found, indicating that marine samples are better represented by ESI than terrestrial samples, which have an abundant portion of hydrophobic poorly ionizable material. Title in thesis list of papers: Investigating the ionisation of dissolved organic matter by electrospray ionisation

The thawing of permafrost is leading to increased export of organic matter into aquatic ecosystems that was previously stored within frozen peatland soils. This organic matter has been found to be reactive to microbial and photochemical processes, so that permafrost thaw is expected to lead to an increased production of greenhouse gases. Being able to predict the fate of these increased loads of terrestrial organic carbon in aquatic systems is therefore important from a climate change perspective. In a previous study we suggest that terrestrial organic compounds susceptible to photodegradation are also prone to adsorb to mineral particles. Whereas photodegradation stimulates CO2 production, adsorption has the potential to remove organic matter from the water column and store it in the sediment. Warming at high latitudes involves both permafrost thaw and glacial melt. Glacial runoff streams often contain high loads of suspended sediment. As these minerogenic particles are transported downstream the aquatic continuum, they can eventually mix with water containing high concentrations of freshly released organic matter, and act as an adsorbent.In order to predict CO2 production from mobilized permafrost organic matter, we need to study the bioavailability of this material before and after alteration by physical and chemical processes such as photodegradation and adsorption to mineral particles. In this study, we compared the effect of adsorption to glacial suspended sediment to that of photodegradation on the dissolved organic matter composition of surface water collected from a thawing peat plateau in northern Sweden. We used optical measurements and mass spectrometry to evaluate changes in the composition of the organic matter and employed a three-month incubation to determine its bioavailability. Initial results from optical measurements indicate that while chromophoric compounds in general were removed by both photodegradation and adsorption, humic-like fluorescent compounds were more susceptible to photodegradation than adsorption. UV-irradiation increased bioavailability of the organic matter, whereas pre-treatment by adsorption to mineral particles slightly decreased bioavailability compared to the control. Results from this study will help advance our understanding of interactive effects between physico-chemical processes and microbial degradation at an increasingly relevant interface where melting permafrost meets glacial meltwaters.

Different processes contribute to the loss or transformation of dissolved organic matter (DOM) and change DOM concentration and composition systematically along the inland water continuum. Substantial efforts have been made to estimate the importance of microbial and photochemical degradation for DOM concentration and composition and, to some extent, also DOM losses by flocculation, whereas the significance of DOM adsorption to inorganic surfaces has received less attention. Hence, knowledge on the possible extent of adsorption, its effect on DOM loads and composition and on where along the aquatic continuum it might be important, is currently limited or lacking altogether. Here we experimentally determine DOM adsorption onto mineral particles in freshwater ecosystems covering a water residence time gradient in boreal landscape Sweden. We hypothesized that adsorption would gradually decrease with increasing water residence time but actually found that DOM is highly susceptible to adsorption throughout the aquatic continuum. Mass spectrometry and fluorescence analysis on DOM suggest that freshly produced aquatic DOM is less susceptible to adsorption than more terrestrial material. Moreover, the percentage DOM adsorbed in the experiments greatly exceeds the actual adsorption taking place in boreal inland waters across all studied systems. These results illustrate the potential impact of mineral erosion, for example, as a result of agriculture, mining or forestry practices, on the availability, transport, and composition of organic carbon in inland waters.

High-resolution mass spectrometry (HRMS) is a powerful tool in the analysis of dissolved organic matter (DOM), but has several key features that must be understood for robust data interpretation. In this chapter, we review recent research that couples HRMS to other analytical techniques such as chromatography, ion mobility and collision-induced fragmentation, and summarize how these techniques have improved our understanding of complex environmental mixtures. We also review visualization and statistical interpretation techniques and discuss the nature of the analytical window and the ways in which the choice of sample preparation and analysis may affect results and conclusions drawn in HRMS analysis of DOM. This work is intended as a reference for aquatic scientists who want to better understand the analytical obstacles presented by complex mixture HRMS analysis as well as some of the recent analytical strategies that have been developed to deal with these problems.

Peatlands are dominant sources of dissolved organic matter (DOM) to boreal inland waters and play important roles in the aquatic carbon cycle. Yet before peat-derived DOM enters aquatic networks, it needs to pass through peat-stream interfaces that are often characterized by transitions from anoxic or hypoxic to oxic conditions. Aeration at these interfaces may trigger processes that impact the DOM pool, and its fate downstream. Here we experimentally assessed how the aeration of iron- and organic-rich mire-waters influences biodegradation, particle-formation, and modification of DOM. In addition, we investigated how suspended peat-derived particles from mires may influence these processes. We found that within 5 days of aeration, 20% of the DOM transformed into particulate organic matter (POM). This removal was likely due to combination of mechanisms including coprecipitation with oxidized iron, aggregation, and DOM-adsorption onto peat-derived particles. Peat-derived particles promoted microbial activity, but biodegradation was a minor loss mechanism of DOM removal. Interestingly, microbial respiration accounted for only half of the oxygen loss, suggesting substantial nonrespiratory oxygen consumption. The differences observed in DOM characteristics between anoxic and aerated treatments suggest that hydrophilic, aromatic DOM coprecipitated with iron oxides in aerated samples, and the corresponding C:N analysis of generated POM revealed that these organic species were nitrogen-poor. Meanwhile, POM formed via adsorption onto peat-derived particles generated from nonaromatic DOM and more nitrogen-rich species. Hence, selective removal of DOM, dissolved iron, and thus oxygen may be important and overlooked processes in mire-dominated headwater systems.

Hydrothermal vent fluids contain thermally modified dissolved organic matter (DOM) originally entrained from sediments and seawater. We hypothesized that in hydrothermal systems DOM molecular composition is modulated by (i) fluid contribution, (ii) thermal decomposition and pH, and (iii) aspects particular to the vent system. Hence, solid phase extracted (SPE) DOM samples collected along the Mid-Atlantic Ridge (MAR) were molecularly characterized via 15 Tesla Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The molecular character of an oceanic DOM sample was also determined before and after thermal (300 °C) decomposition at acidic and neutral pH. Multivariate statistical analysis indicated that DOM composition was strongly influenced by fluid contribution (Mg2+ concentration between 12 and 54 mM), which correlated positively with measured temperature (between 8 and 375 °C). In comparison, pH of the fluids (between 2.5 and 6.9) had a minor influence. Seafloor pressure, used as a theoretical maximum fluid temperature at the seafloor, separated the fluids collected at Menez Gwen from deeper locations, due to the higher abundance of peptide, carboxyl-rich alicyclic (CRAM) and aromatic molecular formulae at Menez Gwen. Compared with seawater DOM, thermally decomposed DOM had on average lower molecular mass, lower O/C ratios, fewer double bond equivalents, and fewer CRAM formulae but higher aromaticity - the same molecular features displayed by MAR hydrothermal fluids. The study provides evidence that thermal reworking plays a major role in shaping DOM mixtures from hydrothermal fluids along the MAR, which partly represent thermally reworked marine DOM that survived hydrothermal circulation.

Iron (Fe) limits primary productivity and nitrogen fixation in large regions of the world's oceans. Hydrothermal supply of Fe to the global deep ocean is extensive; however, most of the previous work has focused on examining high temperature, acidic, focused flow on ridge axes that create "black smoker" plumes. The contribution of other types of venting to the global ocean Fe cycle has received little attention. To thoroughly understand hydrothermal Fe sources to the ocean, different types of vent site must be compared. To examine the role of more diffuse, higher pH sources of venting, a hydrothermal plume above the Von Damm vent field (VDVF) was sampled for Total dissolvable Fe (unfiltered, TDFe), dissolved Fe (< 0.2 mu m, dFe) and soluble Fe (< 0.02 mu m, sFe). Plume particles sampled in situ were characterized using scanning electron microscopy and soft X-ray spectromicroscopy. The VDVF vents emit visibly clear fluids with particulate Fe (TDFe-dFe, > 0.2 mu m) concentrations up to 196 nmol kg(-1) comparable to concentrations measured in black smoker plumes on the Mid-Atlantic Ridge. Colloidal Fe (cFe) and sFe increased as a fraction of TDFe with decreasing TDFe concentration. This increase in the percentage of sFe and cFe within the plume cannot be explained by settling of particulates or mixing with background seawater. The creation of new cFe and sFe within the plume from the breakdown of pFe is required to close the Fe budget. We suggest that the proportional increase in cFe and sFe reflects the entrainment, breakdown and recycling of Fe bearing organic particulates near the vents. Fe plume profiles from the VDVF differ significantly from previous studies of "black smoker" vents where formation of new pFe in the plume decreases the amount of cFe. Formation and removal of Fe-rich colloids and particles will control the amount and physico-chemical composition of dFe supplied to the deep ocean from hydrothermal systems. This study highlights the differences in the stabilization of hydrothermal Fe from an off-axis diffuse source compared to black smokers. Off-axis diffuse venting represent a potentially significant and previously overlooked Fe source to the ocean due to the difficulties in detecting and locating such sites.

This paper investigates the relationship between apparent size distribution and molecular complexity of dissolved organic matter from the natural environment. We used a high pressure size exclusion chromatography (HPSEC) method coupled to UV-Vis diode array detection (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) in order to compare the apparent size of natural organic matter, determined by HPSEC-UV and the molecular mass determined online by ESI-MS. We found that there was a clear discrepancy between the two methods, and found evidence for an important pool of organic matter that has a strong UV absorbance and no ESI-MS signal. Contrary to some previous research, we found no evidence that apparently high molecular weight organic matter is constituted by aggregates of low molecular weight (

Marine dissolved organic matter (DOM) is a large (660 Pg) pool of reduced carbon that is subject to thermal alteration in hydrothermal systems and sedimentary basins. In natural high-temperature hydrothermal systems, DOM is almost completely removed, but the mechanism and temperature dependence of this removal have not been studied to date. We investigated molecular-level changes to DOM that was solid-phase extracted (SPE-DOM) from the deep ocean of the North Pacific Ocean. This complex molecular mixture was experimentally exposed to temperatures between 100 and 380 °C over the course of two weeks in artificial seawater, and was then characterised on a molecular level via ultrahigh-resolution Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS). Almost 93% of SPE-DOM was removed by the treatment at 380 °C, and this removal was accompanied by a consistent pattern of SPE-DOM alteration across the temperatures studied. Higher molecular weight and more oxygen rich compounds were preferentially removed, suggesting that decarboxylation and dehydration of carboxylic acid and alcohol groups are the most rapid degradation mechanisms. Nitrogen containing compounds followed the same overall trends as those containing just C, H and O up to 300 °C. Above this temperature, the most highly altered samples contained very little of the original character of marine DOM, instead being mainly composed of very low intensity N- and S- containing molecules with a high H/C ratio (>1.5). Our results suggest that abiotic hydrothermal alteration of SPE-DOM may already occur at temperatures above 68 °C. Our experiments were conducted without a sedimentary or mineral phase, and demonstrate that profound molecular alteration and almost complete removal of marine SPE-DOM requires nothing more than heating in a seawater matrix.

Natural dissolved organic matter (DOM) is an ultracomplex mixture that is essential to global carbon cycling but is poorly understood because of its complexity. The most powerful tool for the DOM characterization is high-resolution mass spectrometry (HRMS) generally combined to direct infusion (DI) as sample introduction. Liquid chromatography (LC) represents a compelling alternative to DI; however, state-of-the-art techniques involve only offline LC-HRMS approaches, which have important logistical drawbacks that make DOM analysis more challenging. This study introduces a new method based on online coupling of liquid chromatography to high resolution mass spectrometry, able to overcome the disadvantages of usual approaches. It is characterized by high reproducibility (% Bray-Curtis dissimilarity among replicates ≈ 2.5%), and it reduces transient complexity and contaminant interferences, thus increasing the signal-to-noise ratio (S/N), leading to the identification of an overall larger number of formulas in the mixture. Moreover, the application of an in silico fractionation prior to the statistical analysis allows an easy, flexible, fast, and detailed comparison of DOM samples from a variety of sources with a single chromatographic run.

The reactivity continuum (RC) model is a powerful statistical approach for describing the apparent kinetics of bulk organic matter (OM) decomposition. Here, we used ultrahigh resolution mass spectrometry data to evaluate the main premise of the RC model, namely that there is a continuous spectrum of reactivity within bulk OM, where each individual reactive type undergoes exponential decay. We performed a 120 day OM decomposition experiment on lake water, with an untreated control and a treatment preexposed to UV light, and described the loss of bulk dissolved organic carbon with RC modeling. The behavior of individual molecular formulas was described by fitting the single exponential model to the change in peak intensities over time. The range of the empirically derived apparent exponential decay coefficients (k

Hydrothermal venting often occurs at submarine volcanic calderas on island arc chains, typically at shallower depths than mid–ocean ridges. The effect of these systems on ocean biogeochemistry has been under-investigated to date. Here we show that hydrothermal effluent from an island arc caldera was rich in Fe(III) colloids (0.02–0.2 µm; 46% of total Fe), contributing to a fraction of hydrothermal Fe that was stable in ocean water. Iron(III) colloids from island arc calderas may be transferred into surrounding waters (generally 0–1500 m depth) by ocean currents, thereby potentially stimulating surface ocean primary productivity. Hydrothermal Fe oxyhydroxide particles (>0.2 µm) were also pervasive in the studied caldera and contained high concentrations of oxyanions of phosphorus (P), vanadium (V), arsenic (As), and manganese (Mn). Hydrothermal island arcs may be responsible for > 50% of global hydrothermal P scavenging and > 40% V scavenging, despite representing

We investigated the application of the LTQ-Orbitrap mass spectrometer (LTQ-Velos Pro, Thermo Fisher) for resolving complex mixtures of natural aquatic dissolved organic matter (DOM) and compared this technique to the more established state-of-the-art technique, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS, Bruker Daltonics), in terms of the distribution of molecular masses detected and the reproducibility of the results collected. The Orbitrap was capable of excellent reproducibility: Bray-Curtis dissimilarity between duplicate measurements was 2.85 ± 0.42% (mean ± standard deviation). The Orbitrap was also capable of the detection of most major ionizable organic molecules in typical aquatic mixtures, with the exception of most sulfur and phosphorus containing masses. This result signifies that the Orbitrap is an appropriate technique for the investigation of very subtle biogeochemical processing of bulk DOM. The lower costs (purchase and maintenance) and wider availability of Orbitrap mass spectrometers in university departments means that the tools necessary for research into DOM processing at the molecular level should be accessible to a much wider group of scientists than before. The main disadvantage of the technique is that substantially fewer molecular formulas can be resolved from a complex mixture (roughly one third as many), meaning some loss of information. In balance, most biogeochemical studies that aim at molecularly fingerprinting the source of natural DOM could be satisfactorily carried out with Orbitrap mass spectrometry. For more targeted metabolomic studies where individual compounds are traced through natural systems, FTICR-MS remains advantageous.

Hydrothermal vent fluids are highly enriched in iron (Fe) compared to ambient seawater, and organic ligands may play a role in facilitating the transport of some hydrothermal Fe into the open ocean. This is important since Fe is a limiting micronutrient for primary production in large parts of the world's surface ocean. We have investigated the concentration and speciation of Fe in several vent fluid and plume samples from the Nifonea vent field, Coriolis Troughs, New Hebrides Island Arc, South Pacific Ocean using competitive ligand exchange-adsorptive cathodic stripping voltammetry (CLE-AdCSV) with salicylaldoxime (SA) as the artificial ligand. Our results for total dissolved Fe (dFe) in the buoyant hydrothermal plume samples showed concentrations up to 3.86 mu M dFe with only a small fraction between 1.1 and 11.8% being chemically labile. Iron binding ligand concentrations (M) were found in mu M level with strong conditional stability constants up to logK(FeL,Fe)(3+) of 22.9. Within the non-buoyant hydrothermal plume above the Nifonea vent field, up to 84.7% of the available Fe is chemically labile and [L] concentrations up to 97 nM were measured. [L] was consistently in excess of Fe-lab, indicating that all available Fe is being complexed, which in combination with high F-lab values in the non-buoyant plume, signifies that a high fraction of hydrothermal dFe is potentially being transported away from the plume into the surrounding waters, contributing to the global oceanic Fe budget.

Corrigendum to “Thermally altered marine dissolved organic matter in hydrothermal fluids” [Organ. Geochem. 110 (2017) 73–86]

Oceanic dissolved organic carbon (DOC) is an important carbon pool, similar in magnitude to atmospheric CO2, but the fate of its oldest forms is not well understood1, 2. Hot hydrothermal circulation may facilitate the degradation of otherwise un-reactive dissolved organic matter, playing an important role in the long-term global carbon cycle. The oldest, most recalcitrant forms of DOC, which make up most of oceanic DOC, can be recovered by solid-phase extraction. Here we present measurements of solid-phase extractable DOC from samples collected between 2009 and 2013 at seven vent sites in the Atlantic, Pacific and Southern oceans, along with magnesium concentrations, a conservative tracer of water circulation through hydrothermal systems. We find that magnesium and solid-phase extractable DOC concentrations are correlated, suggesting that solid-phase extractable DOC is almost entirely lost from solution through mineralization or deposition during circulation through hydrothermal vents with fluid temperatures of 212–401 °C. In laboratory experiments, where we heated samples to 380 °C for four days, we found a similar removal efficiency. We conclude that thermal degradation alone can account for the loss of solid-phase extractable DOC in natural hydrothermal systems, and that its maximum lifetime is constrained by the timescale of hydrothermal cycling, at about 40 million years3.

Short sediment cores were collected from ~1100 m water depth at the top of Hook Ridge, a submarine volcanic edifice in the Central Basin of the Bransfield Strait, Antarctica, to assess Fe and Mn supply to the water column. Low-temperature hydrothermal fluids advect through these sediments and, in places, subsurface H2S is present at high enough concentrations to support abundant Sclerolinum sp., an infaunal tubeworm that hosts symbiotic thiotrophic bacteria. The water column is fully oxic, and oxygen penetration depths at all sites are 2-5 cmbsf. Pore water Fe and Mn content is high within the subsurface ferruginous zone (max. 565 µmol Fe L-1, >3 to 7 cmbsf) — 14-18 times higher than values measured at a nearby, background site of equivalent water depth. Diffision and advection of pore waters supply significant Fe and Mn to the surface sediment. Sequential extraction of the sediment demonstrates that there is a significant enrichment in a suite of reactive, authigenic Fe minerals in the upper 0-5 cm of sediment at one site characterised by weathered crusts at the seafloor. At a site with only minor authigenic mineral surface enrichment we infer that leakage of pore water Fe and Mn from the sediment leads to enriched total dissolvable Fe and Mn in bottom waters. The largest Eh anomaly observed from our Bransfield Strait survey is associated with the elevated total dissolvable metal content in the water column above this coring site.. We hypothesize that the main mechanism for Fe and Mn efflux from the sediment is breach of the surface oxic layer by the abundant Sclerolinum sp., along with episodic enhancements by physical mixing and resuspension of sediment in this dynamic volcanic environment. We propose that Hook Ridge sediments are an important source of Fe and Mn to the deep waters of the Central Basin in the Bransfield Strait, where concentrations are sustained by the benthic flux, and Fe is stabilised in the water column as either colloidal phases or ligand-bound dissolved species. Entrainment of this water mass into the Drake Passage and thereby the Antarctic Circumpolar Current could provide a significant metal source to this HNLC region of the Southern Ocean if mixing and upwelling occurs before removal of this metal pool to underlying sediments. Sediment-covered volcanic ridges are common within rifted margins and may play a previously overlooked role in the global Fe cycle.

Iron (Fe) binding phases in two hydrothermal plumes in the Southern Ocean were studied using a novel voltammetric technique. This approach, reverse titration–competitive ligand exchange–adsorptive cathodic stripping voltammetry, showed that on average 30±21% of dissolved Fe in the hydrothermal plumes was stabilised by chemically labile binding to ligands. The conditional stability constant (log K?FeL) of the observed complexes was 20.61±0.54 (mean±1 SD) for the two vent sites, intermediate between previous measurements of deep ocean ligands (21.4–23; Kondo et al., 2012) and dissolved weak estuarine ligands (Our results indicate that approximately 7.5% of all hydrothermal Fe was stabilised by complexation with ligands. Furthermore, 47±26% of the dissolved Fe in the plume existed in the colloidal size range (0.02–0.2 µm). Our data suggests that a portion (?7.5%) of hydrothermal Fe is sufficiently stabilised in the dissolved size fraction (

The hydrothermal vents on the East Scotia Ridge are the first to be explored in the Antarctic and are dominated by large peltospiroid gastropods, stalked barnacles (Vulcanolepas sp.) and anomuran crabs (Kiwa sp.) but their food webs are unknown. Vent fluid and macroconsumer samples were collected at three vent sites (E2, E9N and E9S) at distances of tens of metres to hundreds of kilometres apart with contrasting vent fluid chemistries to describe trophic interactions and identify potential carbon fixation pathways using stable isotopes. δ(13)C of dissolved inorganic carbon from vent fluids ranged from -4.6‰ to 0.8‰ at E2 and from -4.4‰ to 1.5‰ at E9. The lowest macroconsumer δ(13)C was observed in peltospiroid gastropods (-30.0‰ to -31.1‰) and indicated carbon fixation via the Calvin-Benson-Bassham (CBB) cycle by endosymbiotic gamma-Proteobacteria. Highest δ(13)C occurred in Kiwa sp. (-19.0‰ to -10.5‰), similar to that of the epibionts sampled from their ventral setae. Kiwa sp. δ(13)C differed among sites, which were attributed to spatial differences in the epibiont community and the relative contribution of carbon fixed via the reductive tricarboxylic acid (rTCA) and CBB cycles assimilated by Kiwa sp. Site differences in carbon fixation pathways were traced into higher trophic levels e.g. a stichasterid asteroid that predates on Kiwa sp. Sponges and anemones at the periphery of E2 assimilated a proportion of epipelagic photosynthetic primary production but this was not observed at E9N. Differences in the δ(13)C and δ(34)S values of vent macroconsumers between E2 and E9 sites suggest the relative contributions of photosynthetic and chemoautotrophic carbon fixation (rTCA v CBB) entering the hydrothermal vent food webs vary between the sites.

Here we demonstrate the use of reverse titration - competitive ligand exchange-adsorptive cathodic stripping voltammetry (RT-CLE-ACSV) for the analysis of iron (Fe) binding ligands in seawater. In contrast to the forward titration, which examines excess ligands in solution, RT-CLE-ACSV examines the existing Fe-ligand complexes by increasing the concentration of added (electroactive) ligand (1-nitroso-2-naphthol) and analysis of the proportion of Fe bound to the added ligand. The data manipulation allows the accurate characterisation of ligands at equal or lower concentrations than Fe in seawater, and disregards electrochemically inert dissolved Fe such as some colloidal phases. The method is thus superior to the forward titration in environments with high Fe and low ligand concentrations or high concentrations of inert Fe. We validated the technique using the siderophore ligand ferrioxamine B, and observed a stability constant [Formula: see text] of 0.74-4.37×10(21) mol(-1), in agreement with previous results. We also successfully analysed samples from coastal waters and a deep ocean hydrothermal plume. Samples from these environments could not be analysed with confidence using the forward titration, highlighting the effectiveness of the RT-CLE-ACSV technique in waters with high concentrations of inert Fe.

In the austral summer of 2011 we undertook an investigation of three volcanic highs in the Central Bransfield Basin, Antarctica, in search of hydrothermal activity and associated fauna to assess changes since previous surveys and to evaluate the extent of hydrothermalism in this basin. At Hook Ridge, a submarine volcanic edifice at the eastern end of the basin, anomalies in water column redox potential (E(h)) were detected close to the seafloor, unaccompanied by temperature or turbidity anomalies, indicating low-temperature hydrothermal discharge. Seepage was manifested as shimmering water emanating from the sediment and from mineralised structures on the seafloor; recognisable vent endemic fauna were not observed. Pore fluids extracted from Hook Ridge sediment were depleted in chloride, sulfate and magnesium by up to 8% relative to seawater, enriched in lithium, boron and calcium, and had a distinct strontium isotope composition ((87)Sr/(86)Sr = 0.708776 at core base) compared with modern seawater ((87)Sr/(86)Sr ≈ 0.70918), indicating advection of hydrothermal fluid through sediment at this site. Biogeochemical zonation of redox active species implies significant moderation of the hydrothermal fluid with in situ diagenetic processes. At Middle Sister, the central ridge of the Three Sisters complex located about 100 km southwest of Hook Ridge, small water column E(h) anomalies were detected but visual observations of the seafloor and pore fluid profiles provided no evidence of active hydrothermal circulation. At The Axe, located about 50 km southwest of Three Sisters, no water column anomalies in E(h), temperature or turbidity were detected. These observations demonstrate that the temperature anomalies observed in previous surveys are episodic features, and suggest that hydrothermal circulation in the Bransfield Strait is ephemeral in nature and therefore may not support vent biota.

High‐resolution mass spectrometry (HRMS) has become a vital tool for dissolved organic matter (DOM) characterization. The upward trend in HRMS analysis of DOM presents challenges in data comparison and interpretation among laboratories operating instruments with differing performance and user operating conditions. It is therefore essential that the community establishes metric ranges and compositional trends for data comparison with reference samples so that data can be robustly compared among research groups. To this end, four identically prepared DOM samples were each measured by 16 laboratories, using 17 commercially purchased instruments, using positive‐ion and negative‐ion mode electrospray ionization (ESI) HRMS analyses. The instruments identified ~1000 common ions in both negative‐ and positive‐ion modes over a wide range of m/z values and chemical space, as determined by van Krevelen diagrams. Calculated metrics of abundance‐weighted average indices (H/C, O/C, aromaticity, and m/z) of the commonly detected ions showed that hydrogen saturation and aromaticity were consistent for each reference sample across the instruments, while average mass and oxygenation were more affected by differences in instrument type and settings. In this paper we present 32 metric values for future benchmarking. The metric values were obtained for the four different parameters from four samples in two ionization modes and can be used in future work to evaluate the performance of HRMS instruments Research Funding: Beatriu de Pinos. Grant Number: 2016‐00215 Division of Chemical, Bioengineering, Environmental, and Transport Systems. Grant Number: 1802388 Engineering and Physical Sciences Research Council. Grant Number: EP/L015307/1 French National FT‐ICR network. Grant Number: FR 3624 CNRS H2020 European Research Council. Grant Number: ERC‐STG 716196 H2020 Marie Skłodowska‐Curie Actions. Grant Number: CHROME‐839709 National Science Foundation. Grant Number: AGS‐1531454 Newton Fund. Grant Number: 275910721 NIH. Grant Number: S10OD018475 Vetenskapsrådet. Grant Number: 2018‐04618