MetaboLights

application/xml

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performed putative compound annotation of MS/MS spectra using SIRIUS (version 6.3.7) and the integrated CANOPUS workflow [1]. SIRIUS predicted molecular formulas from fragmentation spectra, while CANOPUS assigned compound classes according to the ClassyFire ontology. Compound assignments (subclass level) with posterior probabilities ≥ 0.5 were retained for downstream analyses. Refs:[1] Dührkop K, Nothias L, Fleischauer M, Reher R, Ludwig M, Hoffmann MA, Petras D, Gerwick WH, Rousu J, Dorrestein PC, Böcker S. 2021. Systematic classification of unknown metabolites using high-resolution fragmentation mass spectra. Nat Biotechnol 39:462–471. doi:10.1038/s41587-020-0740-8.MetaboLightsPublicLiquid Chromatography MS - negative - reverse phaseUntargeted metabolite analysis was performed as in [1], with modifications to account for instrument optimization. Samples were run on an ultra-high performance liquid chromatography system (Vanquish UHPLC, Thermo Scientific) coupled with an Orbitrap Exploris 120 mass spectrometer (Thermo Scientific). A Waters XSelect Premier HSS T3 column (2.1 x 150 mm, 2.5 µm), equipped with a VanGuard FIT pre-column, was used on the instrument for chromatographic separation at 40 °C. The column was eluted at 0.5 ml/min with a combination of solvents: A) 0.1% formic acid in water and B) 0.1% formic acid in acetonitrile. The chromatographic gradient included the following: 1% B (3 min), 15% B (3-6 min), 50% B (6-10.5 min), 95% B (10.5-15 min), 95% B (16.5 min). The column was washed and re-equilibrated with 1% B (8.5 min) between injections. Individual autosampler injections (15 μl each) were taken for negative ion mode analyses. The electrospray voltage was set to 2600 V for negative mode. Settings for source gases were 50 (sheath), 20 (auxiliary), and 1 (sweep) and these settings are presented in arbitrary units. The heated capillary temperature was 375 °C and the vaporizer temperature was 400 °C. [1] Weber L, Armenteros M, Kido Soule M, Longnecker K, Kujawinski EB, Apprill A. 2020. Front Mar Sci 7:582161. doi:10.3389/fmars.2020.582161.Protist parasite infection alters phytoplankton-derived metabolites and restructures natural bacterial communities.Sean AndersonUniversity of New HampshireGrowth Mediumsolventexometabolomemass spectrometry assaySolid phase extraction (SPE) was performed to concentrate and extract metabolites from filtered plankton cultures using a vacuum manifold. Prior to and after SPE, the culture flasks were weighed to calculate the volume of seawater that passed through the cartridges. Acidified filtrate was passed through acid-washed tubing and HyperSep C18 PPL cartridges (Thermo Scientific) that were pre-conditioned with 6 ml of 100% HPLC-grade methanol and 2-3 rounds of 6 ml ultra-pure Milli-Q using gentle vacuum pressure [1] [2]. After filtrate had passed through (~250 ml per bottle), cartridges were rinsed with 2-3 rounds of Milli-Q, removed from the manifold, wrapped in combusted aluminum foil, and frozen at -80 °C. To elute dissolved organic matter (DOM) from the filters, cartridges were rinsed with four volumes of 0.01 M HCl and eluted into combusted 20 ml amber glass vials using 100% methanol (6 ml per sample). Extracts were stored at -20 °C.  Prior to LC-MS analysis, methanol extracts were nearly dried down via vacuum centrifuge on a Savant SPD1010 SpeedVac Concentrator System (Thermo Scientific). Dried extracts were resuspended in 1 ml of 95:5 v/v Milli-Q water: acetonitrile (ACN) solution with deuterated biotin (final concentration 0.05 mg/ml) and vortexed [3]. For each sample, 625 µl were transferred to a 2 ml amber vial. An aliquot of each sample (5 µl) was also transferred to a ‘pooled’ sample, which was used as a quality control to measure instrument drift over the course of the run. Prepared extracts were stored at -20 °C until being run on the mass spectrometer within 4-6 months.   Refs:[1] Fiore CL, Freeman CJ, Kujawinski EB. 2017. Sponge exhalent seawater contains a unique chemical profile of dissolved organic matter. PeerJ 5:e2870. doi:10.7717/peerj.2870.[2] Kido Soule MC, Longnecker K, Johnson WM, Kujawinski EB. 2015. Environmental metabolomics: analytical strategies. Marine Chemistry 177:1–62. doi:10.1016/j.marchem.2015.04.005.[3] Weber L, Armenteros M, Kido Soule M, Longnecker K, Kujawinski EB, Apprill A. 2020. Front Mar Sci 7:582161. doi:10.3389/fmars.2020.582161.sea waterblankScrippsiella acuminata; Amoebophrya sp.Amoebophrya sp.Scrippsiella acuminatahttps://www.ebi.ac.uk/metabolights/MTBLS11219Philip Place. University of New Hampshire. philip.place@unh.edu.Elizabeth Harvey. University of New Hampshire. elizabeth.harvey@unh.edu.Kelsey Poulson-Ellestad. Roosevelt University. kpoulsonellestad@roosevelt.edu.Sean Anderson. University of Georgia. seanceltics34@gmail.com.Raw files containing LC-MS1 and MS2 spectral data were converted into mzML files, trimmed (cutoff at 19 min), and centroided using msConvert [1]. Peak-picking and feature detection were performed in MZmine (version 4.9.14) using the mzwizard workflow and fine-tuning parameters to optimize settings [2]. Instrument control samples (Milli-Q, acetonitrile, and mobile phase) were subtracted from experimental samples during the spectral processing step and are thus not present in the resulting feature tables. MZmine yielded tables of MS1 features and their peak intensities for each sample, with features defined here as unique combinations of mass-to-charge ratios (m/z) and retention times. Average peak intensity of the experimental media (f/2) blank was subtracted from all other samples at each respective time point. Features were set to zero if they were not present in at least 2/3 (or 1/2) replicates for any given treatment and were removed from the table if not present in any sample after curation. Feature intensity was normalized by filtration volume. Refs:[1] Adusumilli R, Mallick P. 2017. Data converstion with ProteoWizard msConvert. Methods Mol Biol 1550:339–368. doi:10.1007/978-1-4939-6747-6_23.[2] Schmid R, Heuckeroth S, Korf A, Smirnov A, Myers O, Dyrlund TS, Bushuiev R, Murray KJ, Hoffmann N, Lu M, Sarvepalli A. 2023. Integrative analysis of multimodal mass spectrometry data in MZmine 3. Nat Biotechnol 41:447–449. doi:10.1038/s41587-023-01690-2.Experiment dayMaterial sampleReplicateDateseanceltics34@gmail.comAll plankton cultures used in this study were obtained from the Roscoff Culture Collection (RCC) [1]. Host cultures of Scrippsiella acuminata (RCC 1627) were maintained in 0.2 µm sterile-filtered autoclaved seawater that was enriched with f/2 minus silica [2]. Host cultures were transferred into fresh media every 7-10 days to maintain exponential growth. Two strains of Amoebophrya sp. spores (RCC 4390 and 4401) were inoculated every 2-3 days with fresh and exponentially growing S. acuminata at a ratio of 1:1 spore to host by volume. All parasite and host cultures were kept at 18 °C on a 12:12 hour light: dark cycle at 80-100 µmol photon/m^2/s. Cell concentrations of host and spores were measured on a Guava easyCyte HT flow cytometer (Millipore) or a Sony SH800Z sorting flow cytometer (Sony). Fresh parasite spores were filtered through 10-µm mesh to separate out the spores (2-5 µm) from remaining host cells. Spores were added separately (4401 and 4390) into healthy S. acuminata cultures in triplicate 1.2-liter bottles at a ratio of 1:1 spore to host based on cell concentration. Triplicate bottles were also included for spore-only, host-only, and f/2 media to control for changes in metabolites from the infected bottles over time. Bottles were sampled daily for 4 days to capture a single infection cycle. On each day, 250 ml was filtered per bottle through 0.2 µm 47 mm polycarbonate filters and filtrate was acidified to achieve pH of 2-3 by adding 650 µl of 100% HCl. Samples for dissolved organic carbon (DOC; 40 ml) were collected daily via gentle filtration through combusted 0.2 µm GFF filters and stored at -20 °C. Flow cytometry samples (1.8 ml) were run live and also preserved in 1% glutaraldehyde and stored at 4 °C. Infection experiments were conducted separately for parasite strains 4401 (Oct 31-Nov 4) and 4390 (Dec 11-Dec 15), each having their own set of controls. A set of Milli-Q control samples were also collected on day 1 for the 4401 spore experiment. Refs:[1] Vaulot D, Le Gall F, Marie D, Guillou L, Partensky F. 2004. The Roscoff Culture Collection (RCC): a collection dedicated to marine picoplankton. Nova Hedwigia 79:49–70.[2] Guillard RRL. 1975. Culture of phytoplankton for feeding marine invertebrates. In WL Smith and MH Chanley [eds.], Culture of marine invertebrate animals. Springer US 29–60.Metabolomicsultra-performance liquid chromatography-mass spectrometrysea waterpooled quality control sampleMetabolomicsdissolved organic matterblankScrippsiella acuminata; Amoebophrya sp.untargeted analysisparasite hostThermo Scientific Orbitrap Exploris 120Amoebophrya sp.solvent blankGrowth Mediummarine metaboliteScrippsiella acuminataexperimental sampleThermo Scientific Vanquish UHPLC Systemuntargeted metabolitesPhytoplanktonsolventexperimental blankexometabolomeultra-performance liquid chromatography-mass spectrometrysea waterpooled quality control sampleMetabolomicsdissolved organic matterblankScrippsiella acuminata; Amoebophrya sp.untargeted analysisparasite hostThermo Scientific Orbitrap Exploris 120Amoebophrya sp.solvent blankGrowth Mediummarine metaboliteScrippsiella acuminataexperimental sampleThermo Scientific Vanquish UHPLC Systemuntargeted metabolitesPhytoplanktonsolventexperimental blankexometabolomeMS data were collected in the Orbitrap analyzer with a mass resolution of 120,000 FWHM at m/z 200. The automatic gain control (AGC) target was set to standard, the maximum injection time was 55 s, and the scan range was 100-1000 m/z. Data-dependent MS/MS data were acquired in the Orbitrap analyzer using higher energy collisional dissociation (HCD) with a normalized collision energy of 35%. The AGC target value for fragmentation spectra was set to standard and the intensity threshold was 2.5e^4. Precursor selection was performed within the quadrupole with a 1 m/z isolation window. All data were collected in profile mode. Three pooled samples were run throughout to assess instrument performance and drift over the sample run. Experimental samples were run in a random order. Two separate LC-MS runs were conducted for parasite strains 4390 and 4401 to account for separate infection experiments. Data collection for each run began at 1.5 min to account for flushing of the divert valve. Instrument control samples (acetonitrile and mobile phase blanks) were also run for each experiment. In some cases, samples were run twice (v2), which reflected initial instrument optimization.falseStrain level differences in marine microbial parasite-host metabolites and host mortality dynamicsParasitism is a widespread source of mortality among marine microbial communities, with members of the diverse alveolate group Syndiniales being most prevalent and frequently identified in global omics studies. Despite their ubiquity, not much is known about Syndiniales parasite-host infection dynamics, strain-level variability, or biogeochemical impacts of metabolites released via infection and lysis. Here, we performed culture-based experiments to compare the infection of Scrippsiella acuminata by two strains of Syndiniales within the genus Amoebophrya sp. (strains 4401 and 4390). To examine changes in metabolite composition and identify important compounds released over an infection cycle, we collected exudates daily for 4 days from both parasite-host pairings and performed untargeted metabolomics using liquid chromatography-mass spectrometry. Samples were also collected daily for flow cytometry to estimate changes in parasite/host abundances and the percentage of infected hosts. Metabolite composition varied between Amoebophrya strains and exhibited distinct temporal patterns and significant differences between infected vs. host (or spore-only) treatments. Specific compounds were more indicative of host infection, as revealed through differential analysis and log2 fold changes. Physiological differences were also recorded at the strain level, with parasite strain 4401 exhibiting a more traditional infection cycle characterized by rapid host infection (38% infected by day 3), decreased host abundance over time, and the release of new spores on days 3-4. In comparison, strain 4390 had a more extended infection timeline, as evidenced by lower host mortality (12% infected by day 3) over a single infection cycle. This work provides novel insights into the biogeochemistry and physiology of widespread marine microbial parasites and reinforces the importance of considering strain-level infection dynamics that may drive mortality in microbial food webs.2026-10-052024-09-30MTBLS11219