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50eV_GE3_01_4248.cdfftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS203/FILES/37-4 idMsMs 25min 30eV_GE6_01_4261.cdfftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS203/FILES/175-4 Ms 25min _GE2_01_4244.cdfftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS203/FILES/60-4 idMsMs 25min 40eV_GE5_01_4257.cdfftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS203/FILES/36-4 MsMs 25min 50eV_GE4_01_4131.cdfprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS203Not applicable.MetaboLightsPublicmicrOTOF-Q II ESI-Qq-TOF (Bruker)2 µl of the 40% methanol leaf extracts were separated by U-HPLC performed on a Dionex Ultimate 3000 UHPLC (Dionex, Sunnyvale, USA) system with a Acclaim (Thermo Scientific) column, particle size 2.2 µm, 2.1 x 150mm [1]. The following binary gradient was applied at 30 ºC: 1 min, isocratic 90% A (de-ionized water, 0.1% [v/v] acetonitrile and 0.05% formic acid), 10% B (acetonitrile and 0.05% formic acid); 24 min linear at 85% B, 5 min isocratic at 85% B. Flow rate was 0.3 ml/min. Injection volume 2 µl (positive detection mode), 3 µl (negative detection mode). </p> Ref: </br> [1] Gaquerel E, Heiling S, Schoettner M, Zurek G, & Baldwin IT . Development and validation of a liquid chromatography-electrospray ionization-time-of-flight mass spectrometry method for induced changes in Nicotiana attenuata leaves during simulated herbivory. Journal of agricultural and food chemistry, 2010, 58(17), 9418-9427. doi:10.1021/jf1017737. PMID:20701244</br>Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis. 10.1073/ps.1503106112. PMID:26170304Dapeng LiMax planck institute for chemical ecologyrosette leafmass spectrometryFor metabolite profiling, approximately 100 mg of ground leaf tissue was weighed. 1 ml extraction buffer (50 mM acetate buffer, pH 4.8, containing 40% methanol) per 100 mg tissue was added and samples were homogenized in a ball mill (Genogrinder 2000; SPEX CertiPrep) for 45 s at 1 x rate and 250 strokes per min. Homogenized samples were centrifuged at 16,000 x g, 4 °C for 30 min, and supernatants were transferred into 1.5 ml microcentrifuge tubes and re-centrifuged as before. Supernatants of 400 µl were transferred to 2 ml glass vials for mass spectrometry-based metabolomics.Nicotiana attenuatahttps://www.ebi.ac.uk/metabolights/MTBLS203Dapeng Li. Max Planck Institute for Chemical Ecology. Max Planck Institute for Chemical Ecology, Department of Molecular Ecology, Jena, Germany. dli@ice.mpg.de.Ian Baldwin. Max Planck Institute for Chemical Ecology. Max Planck Institute for Chemical Ecology, Department of Molecular Ecology, Jena, Germany. baldwin@ice.mpg.de.Emmanuel Gaquerel. University of Heidelberg,. Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 360, Heidelberg, Germany. emmanuel.gaquerel@cos.uni-heidelberg.de. 06221/ 54-5589.Raw data files were converted to netCDF format using the export function of the Data Analysis v4.0 software (Bruker Daltonics, Bremen, Germany).Collision energydli@ice.mpg.deSeeds from N. attenuata Torrey ex Watson were collected over the last 20 years by Ian T. Baldwin and his collaborators in the Southwestern USA and germinated [1]. The well-characterized inbred line ‘UT’ which we used as a control comparison was collected from southwestern UT in 1996 [2] and has been self-fertilized for 30 generations in glasshouse conditions in Jena, Germany. Metabolic changes induced during Manduca sexta feeding were elicited in a highly synchronized fashion by producing with a fabric pattern wheel three rows of punctures onto each side of the midvein of five fully expanded leaves per plant at rosette stage and immediately applying 1:1 diluted M. sexta oral secretions (OS) to the puncture wounds. Four treated leaves were harvested, pooled, flash-frozen 72 h after elicitation and metabolites were extracted. </p> Ref: </br> [1] Krugel T, Lim M, Gase K, Halitschke R and Baldwin IT. Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology, 2002, 12(4), 117-183. doi:10.1007/PL00012666</br> [2] Glawe GA, Zavala JA, Kessler A, Van Dam NM, & Baldwin IT. Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata. Ecology, (2003), 84(1), 79-90. doi:10.1890/0012-9658(2003)084[0079:ECABCW]2.0.CO;2</br>Metabolomicsultra-performance liquid chromatography-mass spectrometrytandem mass spectrometryNatural variationmetabolomic profilingManduca sextauntargeted metabolitesHerbivoryultra-performance liquid chromatography-mass spectrometrytandem mass spectrometryNatural variationmetabolomic profilingManduca sextauntargeted metabolitesHerbivoryEluted compounds were detected by a high-resolution MicroToF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization source operating in positive ionization mode. </p> Typical instrument settings were as follows: capillary voltage 4500 V, capillary exit 130 V, dry gas temperature 200°C, dry gas flow of 8 l/min. Ions were detected from m/z 50 to 1400 at a repetition rate of 1 Hz. Mass calibration was performed using sodium formate clusters (10 mM solution of NaOH in 50/50% v/v isopropanol/water containing 0.2% formic acid). </p> idMS/MS datasets were acquired by operating the instrument with an isolation mass 400 Da, an isolation width 300 Da (largest width meaning all mass is accessible) and collision energies set either to 20, 30, 40, 50 ev to cover broad range mass signal fragmentation by collision-induced dissociation (CID). idMS/MS data collection was restricted to ten samples that were initially selected based on the high range of chemical variations within the total sample population that these samples captured. 4 µl of each of these samples were first analyzed by UHPLC-ESI/qTOF-MS in profile mode (single MS approach, collision energy of 0 ev) using the aforementioned chromatographic conditions with the objective of identifying compound precursors later on associated with molecular fragments as well as of inferring fragmentation patterns due to in-source collision during ionization (low cone voltage settings). The same samples were then analyzed for idMS/MS data acquisition at 4 different collision energy conditions (20, 30, 40 and 50 ev) separately with all detectable mass signals being equally considered as dissociation targets. This resulted into one MS profile dataset (MS1) and 4 CID-derived MS/MS datasets that were then rapidly aligned and post-processed using open source programs.Natural variation can be extremely useful in unraveling the determinants of phenotypic trait evolution but has rarely been analyzed with unbiased metabolic profiling to understand how its effects are organized at the level of biochemical pathways. Native populations of Nicotiana attenuata, a wild tobacco species, have been shown to be highly genetically diverse for traits important for their interactions with insects. To resolve the chemodiversity existing in these populations, we developed a metabolomics and computational pipeline to annotate leaf metabolic responses to Manduca sexta herbivory. We selected seeds from 43 accessions of different populations from the southwestern United States--including the well-characterized Utah 30th generation inbred accession--and grew 183 plants in the glasshouse for standardized herbivory elicitation. Metabolic profiles were generated from elicited leaves of each plant using a high-throughput ultra HPLC (UHPLC)-quadrupole TOFMS (qTOFMS) method, processed to systematically infer covariation patterns among biochemically related metabolites, as well as unknown ones, and finally assembled to map natural variation. Navigating this map revealed metabolic branch-specific variations that surprisingly only partly overlapped with jasmonate accumulation polymorphisms and deviated from canonical jasmonate signaling. Fragmentation analysis via indiscriminant tandem mass spectrometry (idMS/MS) was conducted with 10 accessions that spanned a large proportion of the variance found in the complete accession dataset, and compound spectra were computationally assembled into spectral similarity networks. The biological information captured by this networking approach facilitates the mining of the mass spectral data of unknowns with high natural variation, as demonstrated by the annotation of a strongly herbivory-inducible phenolic derivative, and can guide pathway analysis.Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis.Li Dapeng D, Baldwin Ian T IT, Gaquerel Emmanuel Emethionine aminopeptidase activity, determination, Tandem, acetylglucosaminyltransferase-like protein, Pflanze, Metabonomic, Mbp1, Metabonomics, sci, Mass Spectrometry Mass Spectrometry, viridiplantae, Animal Grazings, Techniques, exact), Method, insects, Atelocerata, High Performance Liquid Chromatography, HOW, How, 2, AW048865, Elaiosome, myd, l(3)j5D5, WMS, peptidase M activity, 2R)-3-oxo-2-(pent-2Z-enyl)-cyclopentaneacetic acid, 24B, adenomas, rabGAPLP, L-methionine aminopeptidase activity, like-acetylglucosaminyltransferase, Tobacco Hornworm, proportionality to, Herbivories, stru, chemical analysis., Plant Embryo, RabGAP-5, Mbp-1, plants, Mass Spectrometry, l(3)S053606, CG10293, HPLC, l(3)j5B5, High-Performance, Methodological Studies, (1R, Diaspores, RUSC3, Chromatography, tobacco hornworm, High-Performance Liquid, High Speed Liquid, GPHYSD2, Herbivore, SGS, 0904/17, Data Set, gyltl1b-b, land plants, jasmonate signaling, Liquid Chromatography, MYH-associated polyposis, autosomal recessive familial adenomatous polyposis, Maps, Procedure, ACMICD, SZ1, 2R)-3-oxo-2-(2Z)-2-penten-ylcyclopentanacetic acid, MDDGA6, mKIAA0609, Uniramia, Seed, KIAA0609, Manduca sextas, acetylglucosaminyltransferase-like 1A, autosomal recessive, tomato hornworm, Hornworm, fg, RUTBC3, Diaspore, gyltl1b, MS/MS, 2-{(1R, mdc1d, proportionality, Plant, hexapods, Jasmonate, Plant Zygote, rate, Methodological, MASS, Methodological Study, LARGE_HUMAN, 葉 (Japanese, RABGAP5, MDC1D, higher plants, enr, Festa, multiple colorectal, Liquid, anon-EST:Liang-2.39, Procedures, FESTA-L, FESTA-S, P62, FBN, (-)-Jasmonic acid, LARGE1, froggy, Gyltl1a, Hornworms, method, ECTOL1, Grazing, method used in an experiment, Studies, Tobacco, hornblower, Animal, Metabolomic, Manduca sexta, Herbivores, Technique, U19, familial adenomatous polyposis, Grazings, MAP, plantae, BM040, proportion, jasmonate, l(3)s2612, lateral shoot, MS2, MDDGB6, familial adenomatous polyposis 2, 7-epi-jasmonic acid, Zygote, LARGE, 2R)-3-oxo-2-[(Z)-pent-2-enyl]cyclopentyl}acetate, High-Performance Liquid Chromatographies, autosomal recessive multiple colorectal adenomas, OCTD, Study, BPFD#36, DmelCG10293, Zygotes, Ultra Performance Liquid Chromatography, species, UPLC, axillary shoot, Animal Grazing, High-Performance Liquid Chromatography, Elaiosomes, sextas, MUTYH-Associated Polyposis, clone 2.39, Traits, qkr, l(3)S090417, KH93F, Embryos, chemical analysis, MFS1, Insects, tandem MS, WMS2, tobacco hawkmoth, who, Embryo, Manducas, Tobacco Hornworms, 3-oxo-2-(2-pentenyl)cyclopentaneacetic acid, (-)-jasmonic acid, Who/How, Mass Spectrometry-Mass Spectrometry, MYH-Associated Polyposis, Plant Embryos, TRAITS, hoja (Spanish, Insect, tomato hornworm <Manduca sexta>, plan specification, Sphinx sexta, High Pressure, like-glycosyltransferase, Manduca, Plant Zygotes, SSKS, quotient, qkr[93F], wild tobacco, MAP syndrome, High Performance Liquid, assay, Carolina sphinx, FAP2, Tracheata, glycosyltransferase-like protein LARGE1, colorectal adenomatous polyposissecondary metabolite metabolism, secondary metabolite metabolic process, Secondary Metabolisms, Secondary, Animal Grazings, determination, Grazing, Metabolism, Metabolisms, Nicotania tabacum, common tobacco, Herbivories, Nicotiana tabacum var. Samsun, chemical analysis., assay, Animal, secondary metabolism, American tobacco, tobacco, Herbivores, Grazings, Herbivore, Animal GrazingProcedures, methionine aminopeptidase activity, determination, Tandem, FESTA-L, FESTA-S, acetylglucosaminyltransferase-like protein, Pflanze, Metabonomic, Mbp1, FBN, Metabonomics, (-)-Jasmonic acid, Mass Spectrometry Mass Spectrometry, LARGE1, froggy, viridiplantae, Gyltl1a, Hornworms, Animal Grazings, method, Techniques, exact), Method, ECTOL1, insects, Atelocerata, Grazing, method used in an experiment, High Performance Liquid Chromatography, Studies, Tobacco, 2, AW048865, hornblower, Animal, Metabolomic, Manduca sexta, Elaiosome, myd, Trait, Herbivores, Technique, U19, familial adenomatous polyposis, WMS, Grazings, MAP, peptidase M activity, 2R)-3-oxo-2-(pent-2Z-enyl)-cyclopentaneacetic acid, plantae, adenomas, rabGAPLP, BM040, proportion, L-methionine aminopeptidase activity, jasmonate, lateral shoot, like-acetylglucosaminyltransferase, Tobacco Hornworm, proportionality to, MS2, MDDGB6, Herbivories, familial adenomatous polyposis 2, 7-epi-jasmonic acid, chemical analysis., Plant Embryo, RabGAP-5, Mbp-1, Zygote, plants, Mass Spectrometry, LARGE, 2R)-3-oxo-2-[(Z)-pent-2-enyl]cyclopentyl}acetate, High-Performance Liquid Chromatographies, HPLC, autosomal recessive multiple colorectal adenomas, OCTD, Study, BPFD#36, High-Performance, Methodological Studies, (1R, Diaspores, RUSC3, Chromatography, tobacco hornworm, Zygotes, Ultra Performance Liquid Chromatography, High-Performance Liquid, High Speed Liquid, species, UPLC, Southwest U.S., GPHYSD2, axillary shoot, Herbivore, SGS, Animal Grazing, High-Performance Liquid Chromatography, Elaiosomes, sextas, MUTYH-Associated Polyposis, Data Set, gyltl1b-b, Traits, land plants, jasmonate signaling, Liquid Chromatography, MYH-associated polyposis, autosomal recessive familial adenomatous polyposis, Maps, Procedure, ACMICD, 2R)-3-oxo-2-(2Z)-2-penten-ylcyclopentanacetic acid, MDDGA6, Embryos, mKIAA0609, chemical analysis, MFS1, Insects, Uniramia, Seed, KIAA0609, Manduca sextas, tandem MS, acetylglucosaminyltransferase-like 1A, WMS2, tobacco hawkmoth, autosomal recessive, tomato hornworm, Hornworm, fg, Embryo, RUTBC3, Diaspore, gyltl1b, Manducas, Tobacco Hornworms, MS/MS, 2-{(1R, 3-oxo-2-(2-pentenyl)cyclopentaneacetic acid, mdc1d, proportionality, Plant, hexapods, Jasmonate, Plant Zygote, rate, (-)-jasmonic acid, Methodological, Mass Spectrometry-Mass Spectrometry, MASS, MYH-Associated Polyposis, Plant Embryos, Southwest US, Methodological Study, LARGE_HUMAN, TRAITS, hoja (Spanish, 葉 (Japanese, Insect, tomato hornworm <Manduca sexta>, RABGAP5, plan specification, Sphinx sexta, MDC1D, higher plants, High Pressure, like-glycosyltransferase, enr, Manduca, Plant Zygotes, Festa, multiple colorectal, SSKS, quotient, Liquid, wild tobacco, MAP syndrome, High Performance Liquid, assay, Carolina sphinx, FAP2, Tracheata, glycosyltransferase-like protein LARGE1, colorectal adenomatous polyposissecondary metabolite metabolism, secondary metabolite metabolic process, Secondary Metabolisms, Secondary, Animal Grazings, determination, Grazing, Metabolism, Metabolisms, Nicotania tabacum, common tobacco, Herbivories, Nicotiana tabacum var. Samsun, chemical analysis., assay, Animal, secondary metabolism, American tobacco, tobacco, Herbivores, Grazings, Herbivore, Animal GrazingfalseNavigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysisNatural variation can be extremely useful in unraveling the determinants of phenotypic trait evolution but has rarely been analyzed with unbiased metabolic profiling to understand how its effects are organized at the level of biochemical pathways. Native populations of Nicotiana attenuata, a wild tobacco species, have been shown to be highly genetically diverse for traits important for their interactions with insects. To resolve the chemodiversity existing in these populations, we developed a metabolomics and computational pipeline to annotate leaf metabolic responses to Manduca sexta herbivory. We selected seeds from 43 accessions of different populations from the southwestern United States—including the well-characterized Utah 30th generation inbred accession—and grew 183 plants in the glasshouse for standardized herbivory elicitation. Metabolic profiles were generated from elicited leaves of each plant using a high-throughput ultra HPLC (UHPLC)-quadrupole TOFMS (qTOFMS) method, processed to systematically infer covariation patterns among biochemically related metabolites, as well as unknown ones, and finally assembled to map natural variation. Navigating this map revealed metabolic branch-specific variations that surprisingly only partly overlapped with jasmonate accumulation polymorphisms and deviated from canonical jasmonate signaling. Fragmentation analysis via indiscriminant tandem mass spectrometry (idMS/MS) was conducted with 10 accessions that spanned a large proportion of the variance found in the complete accession dataset, and compound spectra were computationally assembled into spectral similarity networks. The biological information captured by this networking approach facilitates the mining of the mass spectral data of unknowns with high natural variation, as demonstrated by the annotation of a strongly herbivory-inducible phenolic derivative, and can guide pathway analysis.2015-06-152015-06-01MTBLS20326170304