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The identification of metabolites in biological samples is challenging due to their chemical and structural diversity. Ion mobility spectrometry (IMS) separates ionized molecules based on their mobility in a carrier buffer gas giving information about the ionic shape by measuring the rotationally averaged collision cross-section (CCS) value. This orthogonal descriptor, in combination with the m / z , isotopic pattern distribution, and MS/MS spectrum, has the potential to improve the identification of molecular molecules in complex mixtures. Urine metabolomics can reveal metabolic differences, which arise as a result of a specific disease or in response to therapeutic intervention. It is, however, complicated by the presence of metabolic breakdown products derived from a wide range of lifestyle and diet-related byproducts, many of which are poorly characterized. In this study, we explore the use of trapped ion mobility spectrometry (TIMS) via LC parallel accumulation with serial fragmentation (PASEF) for urine metabolomics. A total of 362 urine metabolites were characterized from 80 urine samples collected from healthy volunteers using untargeted metabolomics employing HILIC and RP chromatography. Additionally, three analytes (Trp, Phe, and Tyr) were selected for targeted quantification. Both the untargeted and targeted data was highly reproducible and reported CCS measurements for identified metabolites were robust in the presence of the urine matrix. A comparison of CCS values among different laboratories was also conducted, showing less than 1.3% ΔCCS values across different platforms. This is the first report of a human urine metabolite database compiled with CCS values experimentally acquired using an LC-PASEF TIMS-qTOF platform.

Metabolomics based on mass spectrometry can provide quantitative and qualitative information of the pool of metabolites (metabolome) present intracellularly or extracellularly in a given biological system. A typical metabolomics workflow requires several key steps such as quick and robust sample preparations with quenching of metabolism, chemical derivatization if needed, instrumental measurement, data-processing with/without database information and further statistical analysis and interpretation. Here, we introduce general metabolomics workflows for global and targeted analyses using gas chromatography or liquid chromatography coupled with mass spectrometers.

Flow field flow fractionation (FIFFF), inductively coupled plasma-mass spectroscopy (ICP-MS), and transmission electron microscopy (TEM) coupled to X-ray energy dispersive spectrometry (X-EDS) are used in series for the first time to characterize colloids. Results demonstrate the utility of FIFFF-ICP-MS-TEM/X-EDS to relate physical properties (size) of colloids to their chemical properties (chemical composition, surface chemical composition, and colloids-trace elements association). Results suggest that the major part of natural organic matter (NOM) is concentrated in the fraction < 0.01 microm (C2). Aluminum, iron, and manganese are the main colloidal components in the fraction 0.01-0.45 microm (C1). Aluminum occurs as aluminum oxides or aluminosilicates in the whole size range, while iron and manganese occur as individual oxyhydroxides in the size range < 0.20 microm. Within the C2 fraction, Al, Mn, Cu, and Ni elements are complexed to NOM (e.g., humic substances). Iron is complexed to NOM in some samples and probably free in other samples. Lead is totally free in all samples. Within the C1 fraction, Cu and Pb are mostly associated to Fe and Mn oxyhydroxides. Consequently, NOM with Fe and Mn oxyhydroxides are the main colloidal carriers of trace elements in the Loire watershed system.

Warming-induced permafrost thaw could enhance microbial decomposition of previously stored soil organic matter (SOM) to carbon dioxide (CO2) and methane (CH4), one of the most significant potential feedbacks from terrestrial ecosystems to the atmosphere in a changing climate. The environmental parameters regulating microbe-organic matter interactions and greenhouse gas (GHG) emissions in northern permafrost peatlands are however still largely unknown. The objective of this work is to understand controls on SOM degradation and its impact on porewater GHG concentrations across the Stordalen Mire, a thawing peat plateau in Northern Sweden. Here, we applied high-resolution mass spectrometry to characterize SOM molecular composition in peat soil samples from the active layers of a Sphagnum-dominated bog and rich fen sites in the Mire. Microbe-organic matter interactions and porewater GHG concentrations across the thaw gradient were controlled by aboveground vegetation and soil pH. An increasingly high abundance of reduced organic compounds experiencing greater humification rates due to enhanced microbial activity were observed with increasing thaw, in parallel with higher CH4 and CO2 porewater concentrations. Bog SOM however contained more Sphagnum-derived phenolics, simple carbohydrates, and organic- acids. The low degradation of bog SOM by microbial communities, the enhanced SOM transformation by potentially abiotic mechanisms, and the accumulation of simple carbohydrates in the bog sites could be attributed in part to the low pH conditions of the system associated with Sphagnum mosses. We show that Gibbs free energy of C half reactions based on C oxidation state for OM can be used as a quantifiable measure for OM decomposability and quality to enhance current biogeochemical models to predict C decomposition rates. We found a direct association between OM chemical diversity and δ13C-CH4 in peat porewater; where higher substrate diversity was positively correlated with enriched δ13C-CH4 in fen sites. Oxidized sulfur-containing compounds, produced by Sphagnum, were further hypothesized to control GHG emissions by acting as electron acceptors for a sulfate-reducing electron transport chain, inhibiting methanogenesis in peat bogs. These results suggest that warming-induced permafrost thaw might increase organic matter lability, in subset of sites that become wetlands, and shift biogeochemical processes toward faster decomposition with an increasing proportion of carbon released as CH4.

The utility of metabolomics is well documented; however, its full scientific promise has not yet been realized due to multiple technical challenges. These grand challenges include accurate chemical identification of all observable metabolites and the limiting depth-of-coverage of current metabolomics methods. Here, we report a combinatorial solution to aid in both grand challenges using UHPLC-trapped ion mobility spectrometry coupled to tandem mass spectrometry (UHPLC-TIMS-TOF-MS). TIMS offers additional depth-of-coverage through increased peak capacities realized with the multi-dimensional UHPLC-TIMS separations. Metabolite identification confidence is simultaneously enhanced by incorporating orthogonal collision cross section (CCS) data matching. To facilitate metabolite identifications, we created a CCS library of 146 plant natural products. This library was generated using TIMS with N2 drift gas to record the TIMSCCSN2 of plant natural products with a high degree of reproducibility; i.e., average RSD = 0.10%. The robustness of TIMSCCSN2 data matching was tested using authentic standards spiked into complex plant extracts, and the precision of CCS measurements were determined to be independent of matrix affects. The utility of the UHPLC-TIMS-TOF-MS/MS in metabolomics was then demonstrated using extracts from the model legume Medicago truncatula and metabolites were confidently identified based on retention time, accurate mass, molecular formula, and CCS.

Peatlands, which store one third of the terrestrial carbon (C), are subject to large disturbances under a changing climate. It is crucial to understand how microbial and physiochemical factors affect the vulnerability of these large C stores to predict climate‐induced greenhouse gas fluxes. Here, we used a combination of mass spectrometry and spectroscopy techniques, to understand sequential biotic and abiotic degradation pathways of Sphagnum fallax leachate in an anaerobic incubation experiment, in the presence and absence of microorganisms. Removal of microorganisms was carried out by passing aqueous samples through 0.2‐µm filters. Our results revealed that S. fallax leachate degradation by abiotic reactions is a significant contributor to CO2 production. Further, abiotic factors, such as low pH, are responsible for partial dissolved organic carbon (DOC) degradation that produces bioavailable compounds that shift microbial metabolic pathways and stimulate respiration in peat bogs. Acid‐catalyzed hydrolysis of Sphagnum‐ produced glycosides can provide the microbial communities with glucose and stimulate microbial respiration of DOC to CO2. These results, while unique to peatlands, demonstrate the importance and underscore the complexity of sequential abiotic and biotic processes on C cycling in peat bogs. It is therefore crucial to incorporate abiotic degradation and sequential below‐ground biotic and abiotic interactions into climate models for a better prediction of the influence of climate change on DOC stability in peatlands. These findings might not be representative of other ecosystems with different environmental conditions including mineral‐rich peatlands and plant matter in surface peat horizons that comprise discrete microbial populations, and DOC composition. Plain Language Summary: Peatlands store a large amount of carbon (C) in its soil that can be released into the air as CO2. Microorganisms in peatland soil can utilize C and release it into the atmosphere. Abiotic influences (nonmicrobial), such as acidity, can also release C into the atmosphere by complete mineralization and/or by breaking it apart into simpler forms of C for microbial consumption. Here, we incubated dissolved organic carbon leachate from Sphagnum, a dominant vegetation in peatlands, for 70 days and removed microorganisms from one treatment. We found that with no microorganisms, carbon is being released to the air as CO2, and that without microorganisms more CO2 is being released overall. With microorganisms, C is being eaten and transformed differently by microorganisms before being released to the air, which only contributes to ∼10% of the CO2 released in our experiment. We also found that acidity could be cleaving larger forms of C to simpler forms, which these simple forms are more appealing to microorganisms to consume. Therefore, the importance of both abiotic (acidity) and biotic (microorganisms) influences on CO2 production from peatland soils should be considered when predicting ecosystem responses to climate change. Key Points: Abiotic reactions are a significant contributor to CO2 production in acidic peat bogsAcid‐catalyzed hydrolysis of complex molecules (e.g., glycosides) results in enhanced availability of glucose for microbial consumption in peat bogsAbiotic reactions shift microbial metabolic pathways and stimulate respiration in peat bogs [ABSTRACT FROM AUTHOR]

Antibodies play a central role in host immunity by directly inactivating or recognizing an invading pathogen to enhance different immune responses to combat the invader. However, the cellular responses of pathogens to the presence of antibodies are not well-characterized. Here, we used different mass spectrometry techniques to study the cellular responses of the pathogenic fungus Histoplasma capsulatum to monoclonal antibodies (mAb) against HSP60, the surface protein involved in infection. A proteomic analysis of H. capsulatum yeast cells revealed that mAb binding regulates a variety of metabolic and signaling pathways, including fatty acid metabolism, sterol metabolism, MAPK signaling and ubiquitin-mediated proteolysis. The regulation of the fatty acid metabolism was accompanied by increases in the level of polyunsaturated fatty acids, which further augmented the degree of unsaturated lipids in H. capsulatum's membranes and energy storage lipids, such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines and phosphatidylinositols. MAb treatment also regulated sterol metabolism by increasing the levels of cholesterol and ergosterol in the cells. We also showed that global changes in the lipid profiles resulted in an increased susceptibility of H. capsulatum to the ergosterol-targeting drug amphotericin B. Overall, our data showed that mAb induction of global changes in the composition of H. capsulatum membranes can potentially impact antifungal treatment during histoplasmosis. [ABSTRACT FROM AUTHOR]

The utility of metabolomics is well documented; however, its full scientific promise has not yet been realized due to multiple technical challenges. These grand challenges include accurate chemical identification of all observable metabolites and the limiting depth-of-coverage of current metabolomics methods. Here, we report a combinatorial solution to aid in both grand challenges using UHPLC-trapped ion mobility spectrometry coupled to tandem mass spectrometry (UHPLC-TIMS-TOF-MS). TIMS offers additional depth-of-coverage through increased peak capacities realized with the multi-dimensional UHPLC-TIMS separations. Metabolite identification confidence is simultaneously enhanced by incorporating orthogonal collision cross section (CCS) data matching. To facilitate metabolite identifications, we created a CCS library of 146 plant natural products. This library was generated using TIMS with N2 drift gas to record the TIMSCCSN2 of plant natural products with a high degree of reproducibility; i.e., average RSD = 0.10%. The robustness of TIMSCCSN2 data matching was tested using authentic standards spiked into complex plant extracts, and the precision of CCS measurements were determined to be independent of matrix affects. The utility of the UHPLC-TIMS-TOF-MS/MS in metabolomics was then demonstrated using extracts from the model legume Medicago truncatula and metabolites were confidently identified based on retention time, accurate mass, molecular formula, and CCS. [ABSTRACT FROM AUTHOR]