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Samples were run in random order on a Trace GC Ultra mass spectrometer coupled to a ISQ LT and AS3000 II auto sampler (all ThermoScientific) and analyzed as previously described[1]. The electron impact source was set to 70 eV at 280 °C. Resolution was 866 at m/z 502.20 (FWHM = 0.53).
Ref:
[1] Vidoudez C and Pohnert G. Comparative metabolomics of the diatom Skeletonema marinoi in different growth phases. Metabolomics 8, 654-669 (2012). doi:10.1007/s11306-011-0356-6
"],"chromatography_protocol":["Samples were run in random order on a Trace GC Ultra coupled to a ISQ LT and AS3000 II auto sampler (all ThermoScientific) that was equipped with a DB-5ms column (Agilent J&W, 30 m, 0.25 mm internal diameter, 0.25 μm film thickness, 10 m Duraguard pre-column), and analyzed as previously described[1]. A new, deactivated glass liner (ThermoScientific, 5 x 8 x 105 mm inner x outer diameter x length) with glass wool was used for every batch of 21 samples. Samples were injected using split 10 (intracellular) or splitless mode (1 min; extracellular).
Ref:
[1] Vidoudez C and Pohnert G. Comparative metabolomics of the diatom Skeletonema marinoi in different growth phases. Metabolomics 8, 654-669 (2012). doi:10.1007/s11306-011-0356-6
"],"publication":["Metabolomics-derived marker metabolites to characterize\nPhaeocystis pouchetii in natural plankton communities."],"submitter_affiliation":["Friedrich-Schiller University Jena"],"Organism":["Phaeocystis pouchetii"],"technology_type":["mass spectrometry"],"disease":[""],"extraction_protocol":["Samples were thawed and vortexed. For intracellular metabolite profiling, samples (0.5 ml per filter) were transferred into 1.5 ml centrifuge tubes (Eppendorf, Germany). Suspensions were treated for 10 min in an ultrasonic bath, centrifuged (15 min, 30.000 x g, 4°C), and supernatants were transferred to 1.5 ml glass vials. Samples were evaporated to dryness under vacuum before subsequent derivatization.
For extracellular metabolite profiling, aliquots of 1.4 ml of the eluates from solid phase extraction were transferred to 1.5 ml glass vials (A/B: 2 μl internal standard (IS) were added) and evaporated to dryness. Samples were then derivatized with methoxyamine and MSTFA[1]. In cases where the IS peak was missing, samples were re-derivatized with 10 μl MSTFA and incubated for 30 min at 60 °C followed by 30 min at 40 °C.
Ref:
[1] Vidoudez C and Pohnert G. Comparative metabolomics of the diatom Skeletonema marinoi in different growth phases. Metabolomics 8, 654-669 (2012). doi:10.1007/s11306-011-0356-6
"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS758"],"author":["Constanze Kuhlisch. Friedrich-Schiller-University Jena. constanze.kuhlisch@weizmann.ac.il.","Georg Pohnert. Friedrich-Schiller-University Jena. georg.pohnert@uni-jena.de."],"data_transformation_protocol":["Chromatograms were converted to NetCDF with the Xcalibur File Converter (ThermoScientific) and then to RAW with MassLynx 4.1 DataBridge (Waters) to allow background-noise correction with the MassLynx 4.1 CODA tool (Waters, MCQ = 0.8, Smoothing window = 5). Signals were de-convoluted with AMDIS 2.71 (Nist) as one batch job each for intra- and extracellular samples. Peaks were integrated in MET-IDEA 2.08 using the chromatogram with the highest number of components as model ion file. Software settings were as previously descriped[1], instrument type was set to 'quadrupole'.
Ref:
[1] Vidoudez C and Pohnert G. Comparative metabolomics of the diatom Skeletonema marinoi in different growth phases. Metabolomics 8, 654-669 (2012). doi:10.1007/s11306-011-0356-6
"],"study_factor":["Growth phase"],"submitter_email":[""],"sample_collection_protocol":["Phaeocystis pouchetii AJ01 (isolated 1994 from Raunefjord, Norway) was obtained from Aud Larsen (Bergen University, Norway). The strain consists solely of diploid, flagellated cells, and showed no colony formation in culture since isolation[1]. Cultures were grown in sterile-filtered, autoclaved f/2 medium (-Si) based on natural seawater at 6.7 ± 0.9 °C, under a 14:10 h light:dark cycle, at 15-50 μmol photons/s/m^2 provided by fluorescent tubes (Osram L15W/840 Lumilux Cool White). Cultures were shaken once daily. To adapt cells to experimental conditions, the stock culture was transferred in three subsequent passages within the exponential growth phase into fresh medium (10% ν:ν). In 2 l glass bottles, 1.62 l medium (n = 4) was inoculated with 180 mL adapted culture to reach an average initial cell abundance of 4.6 ± 3.3 x 10^3 cells/ml. An additional bottle with 1.8 l medium was used as medium control.
For endometabolite analysis, aliquots of 0.5 L were collected 2 h after onset of light from all cultures and the medium control following 12 days (exponential growth; 'exp'), 26 days (early stationary growth; 'e.stat') and 47 days (late stationary growth; 'l.stat') of culturing. Cells were filtered through GF/C filters (47 mm diameter, Whatman) at 65 kPa under pressure. Wet filters were immediately transferred to 25 ml glass beakers. A single filter was required to pass the entire culture volume from exponential cultures ('exp'), whereas 2 or 3 filters were required to pass the culture volume from early ('e.stat') and late ('l.stat') stationary cultures, respectively. After filtration, cells were immediately re-suspended in 1 ml extraction mix (methanol:ethanol:chloroform, 1:3:1, v: v: v) and, if multiple filters were used, combined in 4 ml glass vials. The time between filtration and metabolite quenching in extraction mix was on average 20 min. As internal standard (IS), 5 μl ('l.stat': 10 μl) aqueous ribitol (4 mM; >99%, Sigma-Aldrich) was added. Samples were kept at -20 °C until analysis 1 month after the end of the experiment.
Ref:
[1] Jacobsen A and Veldhuis MJW. Growth characteristics of flagellated cells of Phaeocystis pouchetii revealed by diel changes in cellular DNA content. Harmful Algae 4, 811-821 (2005). doi:10.1016/j.hal.2004.12.001
"],"omics_type":["Metabolomics"],"study_design":["gas chromatography-mass spectrometry","Arctic food web","algal bloom","biomarker","exometabolome","meta memetabolomics","endometabolome"],"curator_keywords":["gas chromatography-mass spectrometry","Arctic food web","algal bloom","biomarker","exometabolome","endometabolome","meta memetabolomics"],"Organism Part":["Whole Organism"],"metabolite_id_protocol":["Metabolite features (abbreviated as 'met.' and listed in Table S5 of the paper associated with this study) were assigned to 1 of 4 identification levels[1]: metabolites, where both retention index and mass spectrum matched a measured chemical reference standard were classified as identified (level 1). Only these metabolites are mentioned and discussed by their name. Others were ranked according to their spectral similarity to a database compound, and annotated and discussed as putative compound (level 2) or compound class (level 3), or as unidentified (level 4). Chemical reference standards were derivatized as described above. Linear retention indices were calculated, and a match between detected metabolite and reference standard was accepted with ΔRI ≤26[2][3]. Mass spectra as extracted by AMDIS were manually compared to the following spectral databases using MS Search 2.0 g (Nist): NIST 11 library version (mainlib, replib, nist_ri), Golm Metabolome Database libraries T_MSRI_ID (http://csbdb.de/csbdb/dload/dl_msri.html; 2004) and GMD_20111121_VAR5_ALK_MSP (http://gmd.mpimp-golm.mpg.de/download/; 2011), and an in-house database (175 compounds from several metabolite classes including algal extracts of Skeletonema marinoi). Mass spectral annotations were regarded as match with a reverse match factor (R.Match) >900, or tagged with '?' if the factor was 800-900 and '??' if it was 700-800.
Ref:
[1] Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, Daykin CA, Fan TW, Fiehn O, Goodacre R, Griffin JL, Hankemeier T, Hardy N, Harnly J, Higashi R, Kopka J, Lane AN, Lindon JC, Marriott P, Nicholls AW, Reily MD, Thaden JJ, Viant MR. Proposed minimum reporting standards for chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics. 2007 Sep;3(3):211-221. doi:10.1007/s11306-007-0082-2. PMID:24039616
[2] Vandendool H and Kratz PD. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J Chromatogr. 1963 Aug;11:463-71. doi:10.1016/s0021-9673(01)80947-x. PMID:14062605.
[3] Koo I, Shi X, Kim S, Zhang X. iMatch2: compound identification using retention index for analysis of gas chromatography-mass spectrometry data. J Chromatogr A. 2014 Apr 11;1337:202-10. doi:10.1016/j.chroma.2014.02.049. PMID:24630063
"],"metabolite_name":["(ii)(i) Sugar acid","(ii)(i) Monosaccharide","(ii) Hexitol or Monosaccharide (Meox 6TMS)","Xylose (Meox 4TMS)","9,12-Octadecadienoic acid (Me)","(ii) 1-Methyl-6-pyrimidinone","Alanine (2TMS)","Arachidonic acid","(ii) Phytol (3-Eicosyne/ Skel_Cell_071)","(ii)(i) 2-C16:0-Glycerol (2TMS)","Threonine (2TMS)","(ii)(i) Disaccharide","Unknown","(ii) Hexonic acid","(ii) Hexonic acid or Pentonic acid","(ii) Pentafuranose","9,12-Octadecadienoic acid","(ii) 7,10-C16:2-enal","(i) Galactosylglycerol or Glucopyranose","Proline +CO2 (2TMS)","(i) Glucopyranose or Galactinol (9TMS)","(ii) Galacturonic acid (Meox 5TMS)","(ii) Ribitol or Threitol","Phytol","Glycerol","(ii) Hexose-derived","(i) (Di-)Galactosylglycerol","Tetradecanoic acid","(i) 6,9,12-Octadecatrienoic acid (Me) or 7,10,13-Hexadecatrienal","(i) Tricyclo[8.6.0.0(2,9)]hexadeca-3,15-diene or (i) C22:6 (Me or TMS)","(ii) Pentadecanal or Decanediol or Dodecenol","1-Palmitoleoylglycerol (2TMS)","(ii) Glycerol pattern + m/z 285, 333, 375","Phenol","(ii) Hydrocarbon","Erythrose (Meox 3TMS)","Brassicasterol","(ii) 1,6-Anhydroglucose or (alcyl-)Pentofuranose","Hexadecanoic acid","(ii) Threitol","Serine","3-Chloro-1,2-propandiol (2TMS)","alpha-Tocopherol","Ribose (Meox 4TMS)","Pyrrole-2-carboxylic acid (2TMS)","Glycine","Serine (2TMS)","Tetradecanoic acid (Me)","Mannitol","(i) Hexose (TMS)","(i) Maltotriose (Meox 11TMS)","(i) Quinic acid or (ii) Coumaroylquinic acid","(ii) Glycerol","Hexadecanoic acid (Me)","(i) Phytol (3-Eicosyne/ Skel_Cell_071)","Alanine","Crinosterol","Docosahexaenoic acid (Me)","(ii) Hexanoic acid","(i) Pentafuranose or Fructose","6,9,12-Octadecatrienoic acid (Me)","Threonic acid","(ii)(i) Hydrocarbon","(ii) Ergosta-4,6,22-triene or Ergosta-5,22-dien-3-ol (acetate)","Pyroglutamic acid (2TMS) = Glutamate","Glyceraldehyde (Meox 2TMS)","Scyllo-Inositol","(ii) Adenine (N7- 2TMS)","Maltose (Meox TMS)","1,6-Anhydroglucose","Valine","(i) 1,6,11-Dodecatriene","(ii) Octadeca(di)enal","Eicosapentaenoic acid"],"description_synonyms":["acido graso, acidos grasos, Activity, mannite, E-421, Laboratory, postnatal development, Aminosaeure, Metabonomic, Amino acid, D-(-)-Mannitol, Dambose, Mononucleosis, growth and development, Metabonomics, sci, Osmofundin, DmelCG6383, Meat sugar, nitrate(1-), Background, Mesoinositol, i-inositol, Spectrometry-Gas Chromatography, SEA, Cultural, 1, HOW, How, Saturated Fatty Acids, 2, 3, Esterified Fatty Acid, 4, 5, Fs(3)Hor, IKKg, l(3)j5D5, KEY, Key, Esterified, 24B, vegetative growth of a single-celled organism, DAND1, increased, inosite, D-myo-Inositol, ethnicity, DmelCG2684, amino acids, reference sample, developmental field, Gas Liquid Mass Spectrometry, Esterified Fatty, 5R)-Hexane-1, Aliphatic, L-myo-Inositol, stru, crumb, mononucleosis, l(3)S053606, Gas Chromatography Mass Spectrometry, NTef2, Gas Chromatography-Mass, CG10293, free, manna sugar, 2R, l(3)j5B5, Terpen, Aliphatic Acids, dulcite, trioxidonitrate(1-), Gas Mass Spectrometry, glandular fever, Gas-Liquid, Chromatography, Sugar Alcohol, variability of a physical quality, Gammaherpesviral mononucleosis, D1S1733E, Gas-Mass Spectrometry, NO3(-), Isoprenoids, 3R, 3S, 0904/17, Mass Spectrometry Gas Chromatography, (2R, Alcohol, (1r, CG6383, 6S)-cyclohexane-1, mannitol, Aminokarbonsaeure, Disaccharide, Gas-Liquid-Mass Spectrometry, NO3, late, cis-1, Fatty acid, future organ, Fettsaeuren, Spectrum Analysis, Osmitrol, alpha-amino carboxylic acids, 4R, Fs(3)Sz11, Alditols, 6-hexol, DmIKKgamma, Cultural Background, batch, Cultures, dIKK, SZ1, Kenny, precocious, laboratory, trioxonitrate(V), Gas, 5R, Gas-Liquid Chromatography-Mass Spectrometry, 4s, Amino Acid, Acid, E 421, 1L-myo-Inositol, Fatty Acid, SLC20A3, Saturated Fatty Acid, Amino acids, function., growth pattern, 1D-myo-Inositol, nitrate, non-developmental growth, Mass-Gas Chromatography, IKK-gamma, Spectrometry, Aliphatic Acid, 5/4, early, infectious mononucleosis, Terpenoid, fatty acids, meso-Inositol, DmelCG16910, Horka, Isoprenoid, 6-HEXAHYDROXY-CYCLOHEXANE, Filatov's disease, CG2684, Fs(3)Horka, anon-EST:Liang-2.39, Acids, cellular physiological process, l(3)S050920, terpenos, accessory, Mass Spectrometry-Gas Chromatography, Crbs, Saturated Fatty, Pfeiffer's disease (disorder), advanced, Esterified Fatty Acids, Beliefs, 6-cyclohexanehexol, P62, myo-inositol, Aminocarbonsaeure, far, alpha-amino acid, D4H1S1733E, establishment of cell quiescence, supernumerary, dIKK-gamma, organ field, G1/G0 transition, Mono, Crumbs, acides gras, terpene, DmIKK-gamma, E421, Mass, terpeno, Dana, Alditol, field, DmF2, dmIKKgamma, IKK[[gamma]], Metabolomic, Backgrounds, Cyclohexitol, lod, Gammaherpesviral mononucleosis (disorder), study, Fatty Acids, Chiro Inositol, CT19912, 0509/20, l(3)s2612, 6-hexaol, nutrients, 1384/04, D-Mannitol, Spectrometries, myo-Inositol, Cultural Relativisms, Mannitol, (L)-Mannitol, cell growth and/or maintenance, trioxonitrate(1-), IKK, Mass Gas Chromatography, Infectious Mononucleosis, Pfeiffer's disease, Gas-Liquid Chromatography-Mass, Bios I, DmelCG10293, DAN, Customs, culture, statistical analysis, NITRATE ION, Controlled, l(3)j1B5, Sterol, nutrient, Controlling, data, cell physiology, Saturated, Gas Liquid Chromatography Mass Spectrometry, clone 2.39, 5R)-hexane-1, cell cycle quiescence, 5-trans-4, alpha-amino acids, single-organism cellular process, Cultural Backgrounds, qkr, D2L2AD, l(3)S090417, D-mannitol, l(3)07207, Belief, development, Inositol, IKKgamma, GCMS, Fettsaeure, KH93F, CRB, Crb, Terpene, Kissing disease, NB, l(3)S058104, Foods, Mass Spectrometry-Gas, who, terpenes, distinct, Dmikkgamma, increased number, Alcohols, Ins, Chiro-Inositol, postnatal growth, Myoinositol, Terpenoids, Glandular Fever, acide gras, Who/How, Amino, CG16910, Lds, Sugar, present in greater numbers in organism, stationary phase, [NO3](-), Biomasses, Chromatography-Mass Spectrometry, Monocytic angina, qkr[93F], monocytic angina, Relativisms, Glandular fever, Relativism, variable, Cultural Relativism, growth, General activity, hypothesis, CTP"],"name_synonyms":["Metabonomic, Metabonomics, Metabolomic."],"citation_count":["0"],"citation_count_scaled":["0.0"],"download_count_scaled":["0.0"],"normalized_connections":["0.0"],"reanalysis_count_scaled":["0.0"],"view_count_scaled":["0.0"],"additional_accession":[]},"is_claimable":false,"name":"Metabolomics-derived marker metabolites to characterize Phaeocystis pouchetii in natural plankton communities","description":"Phaeocystis pouchetii (Hariot) Lagerheim, 1893 regularly dominates phytoplankton blooms in the Arctic. Through zooplankton grazing and microbial activity, it is considered to be a key resource for the entire marine food web but the actual relevance of biomass transfer to higher trophic levels is still under discussion. Cell physiology and algal nutritional state are suggested to be major factors controlling the observed variability in zooplankton grazing. However, no data have so far yielded insights into the metabolic state of Phaeocystis populations that would allow testing this hypothesis. Therefore, endometabolic markers of different growth phases were determined in laboratory batch cultures using comparative metabolomics and quantified in different phytoplankton blooms in the field. Metabolites, produced during exponential, early and late stationary growth of P. pouchetii were profiled using gas chromatography-mass spectrometry. Then, metabolites were characterized that correlate with the growth phases using multivariate statistical analysis. Free amino acids characterized exponential growth, whereas the early stationary phase was correlated with sugar alcohols, mono- and disaccharides. In the late stationary phase free fatty acids, sterols and terpenes increased. These marker metabolites were then traced in Phaeocystis blooms during a cruise in the Barents Sea and North Norwegian fjords. About 50 endometabolites of P. pouchetii were detected in natural phytoplankton communities. Their relative abundances at Phaeocystis-dominated stations differed from diatom-dominated stations. Mannitol, scyllo- inositol, 24-methylcholesta-5,22-dien-3β-ol, and several free fatty acids were characteristic for Phaeocystis-dominated blooms. Distinct metabolic profiles were detected in the nutrient- depleted community in the inner Porsangerfjord (<0.5 μM NO3-, <0.1 μM PO4-), with high relative amounts of free mono- and disaccharides indicative for a limited culture. This study, therefore, shows how variable physiology of phytoplankton can alter the metabolic landscape of entire plankton communities.
","dates":{"publication":"2020-06-25","submission":"2018-09-27"},"accession":"MTBLS758","cross_references":{"MetaboLights":["MTBLC30997","MTBLC22470","MTBLC33946","MTBLC33942","MTBLC15882","MTBLC15756","MTBLC16449","MTBLC10642","MTBLC3168","MTBLC28661","MTBLC5445","MTBLC26984","MTBLC26986","MTBLC17351","MTBLC18183","MTBLC18721","MTBLC17754","MTBLC18222","MTBLC36006","MTBLC36005","MTBLC36751","MTBLC17306","MTBLC17822","MTBLC17327","MTBLC15843","MTBLC133596","MTBLC15428","MTBLC26271","MTBLC27266","MTBLC28875","MTBLC155905","MTBLC29864"],"ChEBI":["CHEBI:26984","CHEBI:28661","CHEBI:36006","CHEBI:15843","CHEBI:15428","CHEBI:17306","CHEBI:133596","CHEBI:18721","CHEBI:5445","CHEBI:17754","CHEBI:18222","CHEBI:36751","CHEBI:17351","CHEBI:18183","CHEBI:33946","CHEBI:30997","CHEBI:22470","CHEBI:28875","CHEBI:29864","CHEBI:155905","CHEBI:27266","CHEBI:10642","CHEBI:16449","CHEBI:33942","CHEBI:15756","CHEBI:3168","CHEBI:26271","CHEBI:17822","CHEBI:17327","CHEBI:36005","CHEBI:15882","CHEBI:26986"]}}