single-throughput-complementary-high-resolution-analytical-techniques

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Pacific Northwest National Laboratory

We introduce a cheminformatics approach that combines highly selective and orthogonal structure elucidation parameters; accurate mass, MS/MS (MS²), and NMR into a single analysis platform to accurately identify unknown metabolites in untargeted studies. The approach starts with an unknown LC-MS feature, and then combines the experimental MS/MS and NMR information of the unknown to effectively filter out the false positive candidate structures based on their predicted MS/MS and NMR spectra. We demonstrate the approach on a model mixture, and then we identify an uncatalogued secondary metabolite in . The NMR/MS² approach is well suited to the discovery of new metabolites in plant extracts, microbes, soils, dissolved organic matter, food extracts, biofuels, and biomedical samples, facilitating the identification of metabolites that are not present in experimental NMR and MS metabolomics databases.

Structure Elucidation of Unknown Metabolites in Metabolomics by Combined NMR and MS/MS Prediction.

A new MAS-NMR rotor (the WHiMS rotor) has been developed which can reach pressures of 400 bar at 20 °C or 225 bar at 250 °C. These rotors are ideal for mixed phase systems such as a reaction using a solid catalyst with a liquid/supercritical solvent topped with high pressure gas in the head space. After solid and liquid portions of the sample are loaded, the rotor is capped with an o-ring equipped polymer bushing that snaps into a mating groove in the rotor. The bushings incorporate a check valve into the sealing mechanism which allows for pressurization without mechanical manipulation - they will allow gas to flow in but not out. This WHiMS rotor design has enabled experiments on a wide variety of biotic and abiotic mixed-phase systems. Geochemical systems have also been studied, for example, adsorption and confinement studies of supercritical methane/CO in clays and other minerals which display pressure dependent C chemical shifts. Example data from other mixed-phase chemical and microbial systems are reported. These include monitoring metabolite conversion of extremophilic bacteria found in subsurface systems at elevated pressures and real-time operando reactions in catalysis systems - with liquid-quality resolution for H and C NMR spectra.

High temperature/pressure MAS-NMR for the study of dynamic processes in mixed phase systems.

The deep terrestrial biosphere harbours a substantial fraction of Earth's biomass and remains understudied compared with other ecosystems. Deep biosphere life primarily consists of bacteria and archaea, yet knowledge of their co-occurring viruses is poor. Here, we temporally catalogued viral diversity from five deep terrestrial subsurface locations (hydraulically fractured wells), examined virus-host interaction dynamics and experimentally assessed metabolites from cell lysis to better understand viral roles in this ecosystem. We uncovered high viral diversity, rivalling that of peatland soil ecosystems, despite low host diversity. Many viral operational taxonomic units were predicted to infect Halanaerobium, the dominant microorganism in these ecosystems. Examination of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) spacers elucidated lineage-specific virus-host dynamics suggesting active in situ viral predation of Halanaerobium. These dynamics indicate repeated viral encounters and changing viral host range across temporally and geographically distinct shale formations. Laboratory experiments showed that prophage-induced Halanaerobium lysis releases intracellular metabolites that can sustain key fermentative metabolisms, supporting the persistence of microorganisms in this ecosystem. Together, these findings suggest that diverse and active viral populations play critical roles in driving strain-level microbial community development and resource turnover within this deep terrestrial subsurface ecosystem.

Article	Year
Structure Elucidation of Unknown Metabolites in Metabolomics by Combined NMR and MS/MS Prediction. Metabolites\| PubMed ID: 29342073	2018
High temperature/pressure MAS-NMR for the study of dynamic processes in mixed phase systems. Magnetic resonance imaging\| PubMed ID: 30482639	2018
Viruses control dominant bacteria colonizing the terrestrial deep biosphere after hydraulic fracturing. Nature microbiology\| PubMed ID: 30510171	2018

David W. Hoyt

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