Non-targeted liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS) is increasingly applied for the structure-resolved chemical analysis of dissolved organic matter (DOM). With new developments in MS instrumentation and analysis software, the approach has gained substantial momentum over the past decade. However, achieving high-quality analytical data that is reproducible and comparable across laboratories can be a bottleneck in non-targeted metabolomics and organic matter chemical analysis, especially for data reuse in repository-scale analyses. Understanding the capabilities as well as challenges of comparing LC-MS/MS data from different laboratories is necessary for inferring global trends from public data sets. To illuminate instrumentation factors that drive differences and variability, we used a standardized data analysis pipeline, including classical (CMN) and feature-based molecular networking (FBMN), to analyze data from a ring trial by 24 laboratories on identical sample sets of algal and DOM extracts that were mixed in predefined concentrations and spiked with standards. Our results showed that data sets from similar mass spectrometer types with unified instrument parameters were qualitatively comparable, resolving the same general trends and shared mass spectral features. Interlaboratory comparability was best for high-intensity features, while low-intensity features showed greater detection variability. Our analysis also highlights challenges when comparing data from instruments with different acquisition rates or operating with less standardized methods. Lastly, we provide recommendations for data integration, public data sharing, standardization, and best practices for standardized LC-MS/MS data acquisition, which will be critical for long-term time series and intercomparability of DOM chemical analyses.

White-sand forests contribute significantly to dissolved organic matter (DOM) production in the central Amazon, forming blackwater rivers that dominate organic matter export from the Amazon basin to the ocean. Despite their importance in controlling DOM export, white-sand forests are understudied, and it remains unclear whether systematic changes in the formation of blackwater DOM occur and how seasonal variations and extremes like El Niño-associated droughts impact them. We collected soil porewater from two central Amazon white-sand forests for 2 years, spanning a wet La Niña year followed by an El Niño drought year. The molecular composition of DOM was analyzed using high-resolution mass spectrometry, and correlation network analysis was employed to identify ecologically meaningful DOM subsets. Using additional chemical characterization, database annotations, correlation with 14C-age of DOM and climatic variables, and ecological null modeling, we propose five distinct DOM sources: plant litter and throughfall, soil organic matter (SOM) decomposition, root exudation, and two drought response subsets of likely microbial and plant origin. During drought conditions, aboveground plant-derived compounds decreased, while SOM products, root exudates, and drought response compounds increased. These drought responses were qualitatively similar in both years but notably amplified in the drier El Niño year. Drought amplified deterministic control over DOM composition, indicating that DOM reflected directed biological responses and that future droughts are likely to generate similar shifts. Overall, drought substantially altered belowground carbon cycling by shifting DOM sources and inducing stress responses, effects expected to recur and potentially intensify under future climate scenarios.

RATIONALE:Direct infusion mass spectrometry (DI-MS) is a rapid analytical technique widely used in omics research and other fields. However, the complexity of DI-MS spectra frequently leads to co-fragmentation of analytes with similar m/z, resulting in chimeric fragmentation spectra that complicate compound identification. A DI-based tandem mass spectrometric method (DI-MS2), which modulates the intensity of precursors and fragments by the stepwise movement of the quadrupole isolation window, has been shown to successfully deconvolute chimeric fragmentation spectra. Yet, its applicability to different instruments and optimisation has not been evaluated. METHOD:We evaluate the performance of DI-MS2 on two high-resolution instruments: a linear ion trap-Orbitrap (LIT-Orbitrap) and a quadrupole-Orbitrap (Q-Orbitrap). We examined the impact of six instrumental settings, including mass resolving power, isolation window width, step size between MS2 scans, number of microscans, collision energy and automatic gain control (AGC) target, on the analysis of isobaric mixtures with varying m/z differences. RESULTS:The LIT-Orbitrap consistently achieved high-quality chimeric spectra deconvolution with an average similarity score of 0.98 despite unexpected intensity modulation patterns. The Q-Orbitrap provided four times faster measurements but showed more variable results: It achieved a similarity score of 0.96 for isobars with a m/z difference larger than 0.02, but only 0.56 for m/z differences of 0.006. CONCLUSIONS:These findings indicate that the DI-MS2 is a robust and flexible method applicable across different MS platforms, though the Q-Orbitrap may be less suited for highly complex samples with multiple peaks per nominal mass. This highlights the potential of the DI-MS2 for structural elucidation of complex biological mixtures. Additionally, we provide initial setting optimisation guidelines to improve spectra deconvolution and measurement speed.

Soil organic matter (SOM) plays a central role in the global carbon cycle and contributes to storage of C and energy in soils. Farmyard manure (FYM) addition to arable soils is a measure to increase SOM content, microbial activity and abundance of microbial metabolites (e.g., necromass (NM) markers). However, understanding the mechanistic links between soil dynamics and energy storage is hampered due to the chemical complexity of SOM. Non-targeted molecular-level methods like liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FT-ICR-MS) can be used to explore the complex dynamics of SOM, revealing energetic fingerprints and long-term changes in SOM due to FYM addition. We compared water-extractable organic matter (WEOM) from soils of four long-term FYM addition experiments with representative WEOM signatures from maize, bacterial and fungal NM. Long-term FYM addition increased the complexity of WEOM, most pronounced in polar, unsaturated, oxidised and energy-poor compounds. These changes were linked to a 2-3-fold increase in bacterial, plant and fungal NM signatures. Especially bacterial NM in FYM-amended WEOM indicated a shift in dominant energy use channels. Control soils showed a much lower overlap with all NMs, but indicated a higher dominance of fungal energy-use channels, especially for N-containing compounds. A large fraction of WEOM signals (79% in FYM-amended, 94% in control soils) was unrelated to any of the three NM signatures, and was also mainly responsible for the shift in nominal oxidation state of carbon (NOSC) between the fertilisation treatments. LC-FT-ICR-MS provided access to ~600 novel microbial NM markers which are readily soluble and compositionally distinct from classical NM markers (ergosterol, aminosugars, etc.). Overall, we highlight novel insights into NM contribution to SOM by LC-FT-ICR-MS, and how it can assist to constrain compositional and energetic impacts of FYM addition on soils.

Dissolved organic matter (DOM) is one of the most complex chemical mixtures and plays a central role in biogeochemical cycles across our ecosphere. Despite its importance, DOM remains poorly understood at the molecular level. Over the last decades, significant efforts have been made to decipher the chemical composition of DOM by high-resolution mass spectrometry (HRMS) and liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Yet, the complexity and high degree of non-resolved isomers still hamper the full structural analysis of DOM. To overcome this challenge, we adapted a two-dimensional (2D) LC approach consisting of two reversed-phase dimensions with orthogonal pH, followed by MS/MS data acquisition and molecular networking. The 2D chromatography approach mitigates the complexity of DOM, enhancing both the quality of MS/MS spectra and spectral annotation rates. Applying our approach to analyze coastal surface DOM from Southern California (USA), we annotated in total more than 600 structures via MS/MS spectrum matching, which was up to 90% more than in iterative 1D LC-MS/MS analysis with the same total run time. Our data provide an unprecedented view into the molecular composition of coastal DOM, highlighting the potential of 2D LC-MS/MS approaches to decipher ultra-complex mixtures.

A large fraction of organic matter in soils is associated with mineral phases. This association is mediated by manifold processes (reactive transport, drying-rewetting cycles, biofilm formation, digestion of mineral particles, etc.) and ultimately leads to a stabilization of organic matter in soil [1]. Adsorption describes the mechanism for this association at the molecular level. Ultrahigh resolution mass spectrometry like FT-ICR-MS has gained interest in the study of adsorption processes because of its high sensitivity, resolving power and mass accuracy which allow to study the complex organic mixtures present in soils, and therefore provides molecular insight [2]. For that, supernatant composition can be compared before and after adsorption, but this indirect approach is often not sensitive enough to detect the sorption of small amounts of organic matter, i.e., during initial adsorption to pristine mineral surfaces. An alternative approach is to use laser desorption ionization (LDI) to directly analyze the adsorbed molecules on the mineral surfaces. The method theoretically allows laser spot size of ~ 20-50 µm and even allows imaging of thin sections; methodological advances could therefore improve our understanding of soil organic matter and its spatial heterogeneity [3]. We applied LDI-FT-ICR-MS to study the ionization of individual molecules with and without the presence of DOM (SRFA and pine/ beech litter extracts), and measured adsorption isotherms of individual molecular formulas in dissolved organic matter on quartz, illite and goethite after 24h of contact. Analog to organic matrices used in matrix-assisted LDI, detectability of model compounds improved by factor 2.5 – 40 when spiked into a DOM matrix. In case of sinapic acid, presence of DOM shifted the ionization towards the monomer ion [M-H]- as compared to a mixture of mono-, di- [2M-H]- and trimer [3M-H]- species when analyzed in pure form. These results suggest that soil organic matter and its soluble analogs act as suitable matrices in LDI experiments that ensure proper ionization of the mixture as a whole. In a next step, ion abundance data from sorption experiments was used to model the adsorption process of individual molecular formulas by a Langmuir isotherm approach. We derived estimates of sorption capacity and sorption affinity for each molecular formula, and identified the molecular properties explaining differences in both estimates, as well as their differences between mineral phases. Our data highlight the benefits of LDI-FT-ICR-MS for the study of sorption phenomena in soils, and opens perspectives for resolution of spatial heterogeneity in soils. References [1] Kleber, M., Bourg, I. C., ... & Nunan, N. (2021): Dynamic interactions at the mineral–organic matter interface. Nature Reviews Earth & Environment 2: 402-421. [2] Bahureksa, W., Tfaily, M. M., ... & Borch, T. (2021): Soil organic matter characterization by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS): A critical review of sample preparation, analysis, and data interpretation. Environmental Science & Technology 55: 9637-9656. [3] Lohse, M., Haag, R., ... & Lechtenfeld, O. J. (2021). Direct imaging of plant metabolites in the rhizosphere using laser desorption ionization ultra-high resolution mass spectrometry. Frontiers in Plant Science 12: 753812.

Soil organic matter (SOM) plays a central role in the global carbon cycle, influencing for example soil fertility, biodiversity, and erosion. Recent theories predict that SOM is a blend of plant metabolites and their breakdown products, perpetually undergoing recycling and transformation driven by soil organisms such as fungi and bacteria. However, our understanding of SOM remains incomplete due to its complex chemical composition. Particularly, we lack distinct metabolite information for the majority of these compounds or sources, hampering the analysis of SOM structure, it’s genesis, as well as mechanistic understanding of soil processes. Non-targeted analysis by ultrahigh resolution mass spectrometry, foremost Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), has substantially advanced our understanding of organic matter complexity in soils and allows to gain a representative picture through the use of liquid chromatography (LC). To address the above knowledge gaps, we employed LC-FT-ICR-MS for the investigation of three primary types of necromass (maize litter, bacterial and fungal necromass) extracts, as well as aqueous SOM extracts obtained from arable topsoils (2 – 20 cm depth). Water-soluble SOM fractions can be seen as a transition state between higher molecular weight structures in soils like bio- or necromass and its decomposition end products like carbon dioxide or methane. We employed an LC methodology capable of separating dissolved organic matter (DOM) across a broad polarity spectrum, including highly polar compounds that are typically lost during commonly employed solid-phase extraction. Our results show significant differences between farmyard manure-amended (FYM) and unamended (UF) soil DOM according to its nominal carbon oxidation state (NOSC), saturation and molecular mass, that are most prominent for the highly polar fraction of SOM. We assigned intricate markers derived from bacterial, fungal or plant necromass that indicated higher potential necromass contribution to FYM than UF soil DOM, in line with higher microbial activity in these soils. We found that necromass markers contribute most to the CHNO formula class in soil DOM, thereby explaining structural differences between FYM and UF samples. The outcomes of our research represent an initial stride towards the identification of novel molecular markers intrinsic to soil DOM, its thermodynamic properties and N content. In the long term, these techniques will enable not only the detection of shifts in the molecular composition of soil DOM during substrate decomposition but also the recognition of alterations in structural motifs that are associated with specific necromass types. This advancement holds promise for enhancing our understanding of soil DOM dynamics and therefore may hold important implications for soil and ecosystem management.

Polyethylene glycols (PEGs), a major class of water-soluble polymers (WSPs), are widely used in diverse applications which may lead to their release into the environment. This work investigates the reaction of PEGs with photochemically produced hydroxyl radicals (•OH), an important environmental oxidant, and assesses the effect of reaction-induced molecular weight (MW) decreases on PEG biodegradation dynamics in soil and sediment. Probabilistic kinetic modelling revealed a significant reduction in PEG MW after only a few •OH-induced chain scissions on initial PEG molecules. The simulation results were experimentally validated by reacting 13C-labeled PEGs (average MW = 6200 Da) with photochemically produced •OH, resulting in pronounced shifts in the size distribution of PEGs towards lower MWs with increasing reaction extents. Incubations of the initial non-reacted and three incrementally •OH-reacted PEG mixtures over a 150-day period in sediment and soil demonstrated increasing rates and extents of PEG biodegradation to 13CO2 with increasing •OH-reaction extent and thus decreasing PEG average MW. This study underscores the importance of considering the MW distributions of WSPs and their dynamic changes through biotic or abiotic chain scission reactions — showcased herein by reacting PEGs with photochemically produced •OH — in mechanistically understanding WSP biodegradability in natural and engineered receiving environments.

Long-term addition of farmyard manure supports the accumulation of microbial carbon (C) and soil organic matter (SOM), but the effects on energy storage remain unknown. In particular, it remains unresolved whether manure or the stimulation of microbial transformations explains the increased microbial imprint. The latter would suggest that the accumulation of SOM transformation products controls energy storage, rather than manure directly. We hypothesized that the overlap with original manure signatures could be used as a measure of SOM transformation and its effect on SOM's nominal oxidation state of C (NOSC) and energetic potential Delta G0COX. We employed solid-state laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (LDI-FT-ICR-MS) to study molecular signatures of manure samples and topsoil from four long-term field experiments receiving manure, and unfertilized controls. In line with bulk elemental analysis, LDI-FT-ICRMS suggested that manure increased SOM's energetic potential (0.7-1.2 kJ/mol C). Manure addition changed SOM composition by 3-16% of total ion abundance as compared to controls, being larger in longer-running field experiments. Markers unrelated to original manure signatures (i.e., indirect effects) explained 67-84% of molecular changes while markers directly related to manure explained only 2-12%. Long-term manure addition resulted in increased saturation, oxidation and molecular weight, and decreased aromaticity of SOM as compared to unfertilized soils. Accumulated molecules had higher energetic potentials and were, despite being chemically similar to original manure, elevated in mass, suggesting potential use of manure-derived building blocks for microbial synthesis of larger molecules. Molecules with lower energetic potential disappeared in manured samples, mirrored by a higher oxidation state of water-extractable organic matter, pointing to an increased solubility of SOM. Our results indicate a uniform shift in SOM properties upon manure addition, but highlight the role of site-specific trajectories of SOM compositional change. We discuss the implications of manure-induced microbial transformations for energy storage and long-term stability of SOM.

The interactions between plants and soil microorganisms are fundamental for ecosystem functioning. However, it remains unclear if seasonality of plant growth impacts plant-microbial interactions, such as by inducing shifts in the microbial community composition, their biomass, or changes in the microbial uptake of plant-derived carbon. Here, we investigated the stability of the microbial community and their net assimilation of plant-derived carbon over an entire growing season. Using a C3–C4 vegetation change experiment, and taking advantage of a natural 13C label, we measured the plant-derived carbon in lipid biomarkers of soil microorganisms in rhizosphere and bulk soil in two soils with contrasting textures. We found that temporal stability was higher in bacterial than in fungal biomass, whereas the spatial stability of the fungal biomass was higher than that of bacterial biomass. Moreover, symbiotic AM fungi tended to be more stable in the uptake of plant-derived carbon than bacteria and saprophytic fungi. While soil texture did influence microbial community composition as expected, it had no effect on the microbial plant carbon assimilation and the differences between rhizosphere and bulk soil. In addition, the putative differences in carbon utilization between microbial groups, with the exception of AM fungi, were generally smaller than expected, reflecting opportunistic utilization of energy sources. Our results suggest that microbial uptake of plant carbon is primarily limited by plant carbon allocation rather than by environmental factors such as soil texture and seasonality. This indicates that the ongoing carbon assimilation during the growing season is supported by a functional redundancy within the microbial community, which, in turn, helps sustain ecosystem functioning.

Dissolved organic matter (DOM) is an ultracomplex mixture that plays a central role in global biogeochemical cycles. Despite its importance, DOM remains poorly understood at the molecular level. Over the last decades, significant efforts have been made to decipher the chemical composition of DOM by high-resolution mass spectrometry (HR-MS) and liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Yet, the complexity and high degree of nonresolved isomers still hamper the full structural analysis of DOM. To address this challenge, we developed an offline two-dimensional (2D) LC approach using two reversed-phase dimensions with orthogonal pH levels, followed by MS/MS data acquisition and molecular networking. 2D-LC-MS/MS reduced the complexity of DOM, enhancing the quality of MS/MS spectra and increasing spectral annotation rates. Applying our approach to analyze coastal-surface DOM from Southern California (USA) and open-ocean DOM from the central North Pacific (Hawaii), we annotated in total more than 600 structures via MS/MS spectrum matching, which was up to 90% more than that in iterative 1D LC-MS/MS analysis with the same total run time. Our data offer unprecedented insights into the molecular composition of marine DOM and highlight the potential of 2D-LC-MS/MS approaches to decipher the chemical composition of ultracomplex samples.

In the nutrient-poor soils of the Amazon rainforest, phosphorus (P) emerges as a critical limiting factor for ecosystem productivity. Despite these limitations, the Amazon exhibits remarkable productivity that is maintained by its efficient nutrient recycling mechanisms. Central to this process is the role of organic matter, particularly its dissolved (DOM) fraction, which serves as a crucial nutrient reservoir for both plants and microorganisms. This study delves into the dynamics of nutrient-containing DOM within the soils of two contrasting rainforest ecosystems: clayey terra firme forests, known for their robust nutrient recycling and presumed P-limitation, and sandy white-sand forests, characterized by reduced nutrient recycling capacity and presumed nitrogen (N)-limitation. Utilizing ultra-high resolution mass spectrometry (HR-MS), we analyzed the molecular composition of dissolved organic nutrient species. We evidenced nutrient limitation applying innovative concepts: (1) assessing nutrient depletion in DOM via nutrient-to-carbon ratios, (2) comparing the composition of nutrient-enriched DOM pools across soil depth profiles to infer microbial nutrient processing, and (3) examining the temporal variability of nutrient-containing DOM as an indicator of nutrient uptake and production. Our results corroborate the hypothesis of P-limitation in terra firme forests, with significant processing of N-containing DOM also observed, indicating a synergistic demand for both P and N. Surprisingly, white-sand soils exhibited no signs of N-limitation but instead sulfur (S)-limitation, a novel finding for these ecosystems. This study highlights the diversity of potential nutrient limitations in the central Amazon and the importance of the bioavailable “black box” DOM for tropical nutrient cycles.

The interactions between plants and soil microorganisms are fundamental for ecosystem functioning. However, it remains unclear if seasonality of plant growth impacts plant-microbial interactions, such as by inducing shifts in the microbial community composition, their biomass, or changes in the microbial uptake of plant-derived carbon. Here, we investigate the stability of microbial biomass of different functional groups and their net assimilation of plant-derived carbon over an entire growing season. Using a C3-C4 vegetation change experiment, and taking advantage of natural abundances of 13 C, we measured the plant-derived carbon in lipid biomarkers of soil microorganisms in rhizosphere and non-rhizosphere soil. We found that temporal and spatial stability was higher in bacterial than in fungal biomass, while the high temporal stability of all bacterial groups even increased in close proximity to roots. Moreover, differences in the association to plants, i.e., symbionts vs. free-living microorganisms, tend to determine the stability in the uptake of plant-derived carbon. Our results indicate, the inputs of plant-derived carbon over the growing season did not result in a shift in the microbial community composition, but instead, functional groups that are not in obligate symbiosis with plants showed a varying use of soil- and plant-derived carbon.

Soil organic matter (SOM) is an important driver of the global carbon cycle and plays a central role for the fertility, biodiversity, erosion and other key dynamics in soils. However, our understanding of SOM is still hampered by its chemical complexity. SOM is derived from a mixture of plant metabolites and their decomposition products that are continuously recycled and transformed through the action of soil organisms such as fungi and bacteria. This ultimately leads to the build-up of complex organic mixtures of plant, fungal and bacterial biomass as well as their respective decomposition products (necromass), which lack distinct metabolite information. Ultrahigh resolution mass spectrometry, such as FT-ICR-MS, has contributed much to our understanding of organic matter complexity in soils, but novel methods are needed to identify and trace molecular markers for necromass contribution. To overcome this gap, we studied three major necromass types (maize litter, bacterial and fungal necromass extracts) and arable (top-)soil aqueous SOM extracts via liquid chromatography (LC) and tandem mass spectrometry (MS/MS) coupled with FT-ICR-MS. Our main goal was to find and characterize indicative molecules (via LC-MS) and structural motifs (from MS/MS) for each necromass type, and to detect these within SOM extracts. To this end we applied an LC method that allows the separation of salts from DOM and covers a broad polarity range, including highly polar compounds that are usually lost during solid-phase extraction. MS/MS experiments were conducted on preselected features (data-independent acquisition) in positive and negative ESI modes. We further tested the sensitivity of our method by spike experiments. Our findings represent a first step in uncovering novel molecular markers that are tracked in ongoing decomposition experiments. In the long run, these techniques will allow to not only detect changes in molecular composition of DOM during substrate decomposition, but also to detect changes in structural motifs that are linked to defined necromass types.

<p>The variability of the molecular composition of dissolved organic matter (DOM) in soils is predominantly explained by microbial mineralization and assimilation as well as interactions with mineral surfaces and soil organic matter (SOM). From a temperate site with calcareous soil it is established that the molecular composition of DOM shifts from plant-derived towards microorganism-derived signals in soil depth profiles. This variability is largely explained by microbial activity and only to a minor degree by soil-derived parameters like texture and SOM. Here we analyze the molecular composition of DOM in depth profiles of tropical rainforest soils in order to compare its variability to the temperate site as the tropical ecosystems have larger variability in soil texture and mineralogy and lower SOM content. We collected porewater in soil profiles of four ecosystems belonging to two major Amazon rainforest types, <em>terra firme</em> forest on clay soils and white-sand forest on sandy soils. We analyzed the molecular composition of DOM using solid-phase extraction and ultrahigh resolution mass spectrometry.</p> <p>The concentration and composition of DOM differed strongly between the sandy white-sand and clayey <em>terra firme</em> sites. DOM concentration in white-sand soils was much higher and decreased less with depth compared to the <em>terra firme</em> forests. Topsoil DOM in white-sand forests was characterized by high abundance of aromatic plant-derived compounds, whereas it reflected a stronger microbial imprint in <em>terra firme</em> sites. The molecular composition of DOM changed significantly with depth at all sites. In both <em>terra firme</em> and one white-sand forest the transformation was consistent with the expected shift from plant-derived signals towards increasing microbial reworking. The Bray-Curtis dissimilarity between topsoil and greater depth was considerably higher in <em>terra firme</em> compared to white-sand soils with values of 0.32 +- 0.06 and 0.14 +- 0.04, respectively, suggesting much slower DOM transformation in sandy soils. The dissimilarity was correlated similarly to pH, clay content and mineralogy (Pearson R<sup>2 </sup>= 0.27, 0.21 and 0.23, respectively). The high dissimilarity in the <em>terra firme </em>sites that vary strongly in clay content and mineralogy was only significantly correlated to pH (R<sup>2</sup> = 0.16). This suggests that clay content and mineralogy, likely linked to DOM adsorption, were less important for the observed depth trend, which aligns with results from the temperate site. The significant effect of soil pH on the molecular transformation is in line with the importance of microorganisms for DOM transformation as soil pH is a major control on microbial community structure. Overall, our results suggest that similar processes control DOM transformation in temperate and tropical ecosystems, which are likely linked to microbial processing and formation of DOM.</p>

<p>Although representing only a small portion of all the organic matter being present in soils, dissolved organic matter (DOM) is its most mobile and reactive fraction. Along its transport down the soil profile, DOM interacts with minerals via sorption and aggregation phenomena, and with organisms via uptake, exudation or internal recycling. During this downward passage DOM composition changes too, shifting from a dominant plant/ plant decomposition product character to a more decomposer-driven signature. Such trends have been documented well by ultrahigh resolution mass spectrometry (FT-MS) which reveals thousands of molecular compositions (formulas) in one DOM sample (Roth et al. 2019). However, complementary properties of the molecules that constitute these signatures are largely missing. To test if the downward trend in molecular composition would also affect DOM’s fragmentation sensitivity, and if these effects could obscure the final ecological interpretation, we obtained direct injection FT-MS data with and without source-induced fragmentation (SID) at 45 eV. As a test dataset, we used samples from suction plates installed in three soil profiles (at 5, 10, 20, 30 and 60 cm depth) developed on aeolian sand deposits (podzols, cambisols) in Linde, Brandenburg (Germany). These three sites vary mainly in vegetation cover (grassland, oak, pine). All sites showed a clear depth trend as reported earlier, especially without SID turned on. SID clearly decreased the ion abundance of signals in the molecular weight range 300-500 Da (center ~350), while signals in the range 150-400 Da (center ~ 225) increased; more formulas were detected with SID on. With depth, the population of molecules decreasing/ increasing changed significantly, especially between 30 and 60 cm depth, across all sites. At 60 cm depth, decreasing formulas were more aliphatic while increasing formulas were more confined to a narrow area in the center of the van Krevelen space (“island of stability”). Interestingly, the formulas decreasing significantly across all sites were largely CHO (no other heteroatoms, at all depths), S-containing (at 5, 10, 20 and 30 cm) or P-containing (30 and 60 cm), while formulas increasing were CHO (at all depths) and N-containing ones (at all depths, but mainly at 20 and 30 cm), thereby indicating depth-dependent differences and ionization of new N-containing molecules. In ordination space, sites were clearly differentiated according to SID status, depth and site (in this order), but SID did not affect the separation in terms of depth or site, i.e., the ecological interpretation of DOM fingerprints remained similar independent of the SID status. This means that trends in soil DOM studies are likely not obscured by differences in fragmentation during the electrospray ionization process.</p> <p>Reference: Roth, V.-N., Lange, M., Simon, C., Hertkorn, N., Bucher, S., Goodall, T., Griffiths, R. I., Mellado-Vázquez, P. G., Mommer, L., Oram, N. J., Weigelt, A., Dittmar, T., Gleixner, G. (2019): Persistence of dissolved organic matter explained by molecular changes during its passage through soil. <em>Nat. Geosci.</em> <strong>12</strong>: 755–761.</p>

<p>Soil organic matter<strong> </strong>plays important roles in soil reactivity and fertility as well as soil physics. Nevertheless, we know relatively little about the individual molecules that make up soil organic matter but ultimately determine its properties. Ultrahigh-resolution mass spectrometry like FT-ICR-MS has revealed an enormous molecular diversity yet it often remains limited to the water-soluble fractions (i.e., dissolved organic matter) analyzed with electrospray ionization (ESI) that represent only a small fraction of the total organic matter contained in soils. To extend the analytical window and leverage the value of non-targeted mass spectrometry, parallel analyses of soluble (via ESI) and particle-associated organic matter (PAOM) via laser-desorption ionization (LDI) and FT-ICR-MS detection is a promising approach, that has yet to prove its full potential. Here, we studied the sensitivity and robustness of the LDI technique based on a combination of dried arable soils, their aqueous DOM extracts, reference DOM samples (Suwannee River Fulvic Acid, SRFA), model compounds (syringic acid, sinapic acid, syringaldehyde, vanillic acid and tannic acid) and model mineral phases (goethite, illite). DOM samples were used to study the effects of a mineral matrix and dilution, while model compounds and SRFA were used to test the effects of laser strength and presence of an organic matrix on intact ionization of analytes. Lastly, non-extracted and extracted soil samples were used to assess if DOM composition trends observed in solution are reproduced in PAOM composition. In general, ESI ionized a very different fraction of the DOM mixture, being more polar and more saturated, while LDI ionized rather small, low-to-mid polar, and less saturated ions. Besides clear differences in PAOM and DOM analytical windows, molecular trends such as aromaticity or nominal oxidation state were well-aligned. Although most insight was gained by combining both types of analyses, our results therefore suggest that direct analysis of soil particles is a fast, reproducible, sensitive and less invasive alternative to routine protocols employing FT-ICR-MS detection, and avoids additional extraction or purification steps.</p>

Ultrahigh-resolution Fourier transform mass spectrometry (FTMS) has revealed unprecedented details of natural complex mixtures such as dissolved organic matter (DOM) on a molecular formula level, but we lack approaches to access the underlying structural complexity. We here explore the hypothesis that every DOM precursor ion is potentially linked with all emerging product ions in FTMS2 experiments. The resulting mass difference (Δm) matrix is deconvoluted to isolate individual precursor ion Δm profiles and matched with structural information, which was derived from 42 Δm features from 14 in-house reference compounds and a global set of 11 477 Δm features with assigned structure specificities, using a dataset of ∼18 000 unique structures. We show that Δm matching is highly sensitive in predicting potential precursor ion identities in terms of molecular and structural composition. Additionally, the approach identified unresolved precursor ions and missing elements in molecular formula annotation (P, Cl, F). Our study provides first results on how Δm matching refines structural annotations in van Krevelen space but simultaneously demonstrates the wide overlap between potential structural classes. We show that this effect is likely driven by chemodiversity and offers an explanation for the observed ubiquitous presence of molecules in the center of the van Krevelen space. Our promising first results suggest that Δm matching can both unfold the structural information encrypted in DOM and assess the quality of FTMS-derived molecular formulas of complex mixtures in general.

Tropical rivers such as the Rio Negro constitute a major portion of the global aquatic flux of dissolved organic carbon (DOC) entering the ocean, but the exact amount, source contributions and fate of terrestrial DOC remain unknown. We investigated the role of valley and upland whitesand ecosystems (WSEs) and terra firme plateaus in forming blackwater tributaries in the Rio Negro basin to develop novel constraints for the terrestrial export of carbon. 5709 molecular markers from ground- and surface waters of two contrasting valley and upland sites feeding Rio Negro tributaries were identified by ultrahigh resolution mass spectrometry (FT-MS), analyzed by multivariate statistics and compared to known Rio Negro markers. In a Principal Coordinates Analysis, valley and upland DOC molecular composition differed by 78% from plateau DOC, which was characterized by reworked, aliphatic and unsaturated N- and S-containing molecules, while valley and upland DOC contained mainly condensed aromatics, aromatics and oxidized unsaturated structures. Valley and upland samples differed by 10% in molecular DOC composition and by their isotopic content (14C of SPE-DOC, 18O and 2H of water) which indicated differences in hydrology and C turnover. Against expectation, markers of widespread whitesand valleys did not emerge as a major source of Rio Negro markers, but specific upland markers did. Pubchem suggested chromene and benzofuran structures as promising candidates for further study. Our findings indicate that the export of molecular markers diverges from expected transport-limited DOC behavior, and thereby opens new avenues for source annotation beyond DOC quantity. Terrestrial DOC from upland whitesand areas is a major source of specific blackwater molecules missing in the regional ecosystem C balance, whereas C export from the whitesand valleys and especially from terra firme plateaus represents mainly recycled and transformed carbon not directly affecting the ecosystem C balance and possibly, the watersheds downstream molecular signature. Our study highlights the potential of high-resolution techniques to constrain carbon balances of ecosystems and landscapes by novel molecular markers. A comparison with other terrestrial DOM datasets indicated molecular similarities with temperate acidic soils and tropical rivers that warrant further analysis of common DOM markers. Implications, limitations, and future challenges are discussed in the light of potential applications of diagnostic molecular links for DOC source annotation and estimation of terrestrial DOM export in the land-to-ocean continuum.