MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/m_MTBLS13756_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/m_MTBLS13756_LC-MS_negative_reverse-phase_metabolite_profiling-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/m_MTBLS13756_LC-MS_negative_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/m_MTBLS13756_LC-MS_positive_reverse-phase_metabolite_profiling-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/a_MTBLS13756_LC-MS_negative_reverse-phase_metabolite_profiling-1.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/a_MTBLS13756_LC-MS_positive_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/a_MTBLS13756_LC-MS_positive_reverse-phase_metabolite_profiling-1.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/a_MTBLS13756_LC-MS_negative_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756/s_MTBLS13756.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13756For extant HGL-DTG annotation and references, see (Heiling et al., 2016). The annotation process in this study consisted of the following steps: confirming the presence of the aglycone in ESI(+) measurements, identifying the [M+NH4]+ adduct in ESI(+) mode, manually identifying within chromatogram files the number of isomers separated by our chromatography and comparing m/z and retention times, i.e., the analysis of the chromatographic retention sequence, to described HGL-DTGs in (Heiling et al., 2016) (cf. Supplementary Tables S2 and S6). In ESI(+) mode, HGL-DTG mass spectra typically exhibited the aglycone mass fragment of m/z 271.24 without all sugar moieties and hydroxyl groups, as described before (Heiling et al., 2016). The DTGs were annotated by the presence of the aglycone mass fragment m/z 271.24 in ESI(+) mode co-occurring at the same timepoint with a [M+NH4]+ adduct using extracted ion chromatograms (EIC). The presence of the aglycone mass was additionally verified by independently measured MS/MS spectra, if obtained by the automated peak picking ddMS2. If present, the [M-H]− mass feature of the ESI(-) mode was used for relative quantification due to only marginal fragmentation in this mode. The [M+H]+ ion was barely detectable by ESI(+) and was only available in rare cases. Use of the adduct mass feature [M+NH4]+ for quantification was assumed to be less accurate than the [M-H]− as the ESI(+) mode caused extensive in-source fragmentation of HGL-DTGs.MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phaseReversed-phase measurements were performed with a Waters Acquity UPLC system and a C18 column (100 mm × 2.1 mm containing 1.7 μm diameter particles, Waters) connected to an Exactive Orbitrap-type MS or Exactive Orbitrap-focus (Thermo Fisher Scientific, Waltham, MA, USA) for MS/MS measurements. The injection volume was 3 μL. The 20 min chromatography was executed with a stable flow of 0.4 mL/min. Solvents were A, water, and B, acetonitrile, both containing 0.1% formic acid. Column temperature was constantly at 40 °C. Starting with 99% A for 1 min, a linear gradient was set to 60% A until 11 min, and from then on to 30% A until 13 min. Finally, a linear gradient flush up to 99% B was programmed until 15 min and held for 1 min before returning to 99% A within 1 min. 99% A was held for the remaining chromatography time to equilibrate to start settings.Detailed Profiling of 17-Hydroxygeranyllinalool Diterpene Glycosides from Nicotiana Species Reveals Complex Reaction Networks of Conjugation Isomers. 10.3390/metabo14100562. PMID:39452943Alina EbertMax Planck Institute of Molecular Plant Physiologyshootmass spectrometry assayFrozen samples of 50 mg (±3 mg) fresh weight (FW) were extracted in a mixture of 350:400:200 (v/v/v) methanol:water:chloroform. A 300 μL aliquot was transferred to new plastic tubes, dried in a vacuum concentrator at room temperature overnight and stored at −20 °C before measurement. The extraction procedure consisted of the addition of pre-cooled (−20 °C) methanol, including the internal standard to the frozen plant material, heating of the extracts at 70 °C for 15 min while shaking, the addition of chloroform, heating at 37 °C for 5 min while shaking, the addition of water and final centrifugation for 5 min at 14,000 rpm at room temperature in a benchtop centrifuge. Dried extracts of each replicate were re-dissolved in 200 μL water before routine LC-MS measurements. Extracts for LC-MS/MS measurements were re-dissolved in 50 μL 50% (v/v) methanol:water mixture; for the latter, multiple random replicates of the same species were pooled to obtain a representative sample.Three non-sample controls ('blanks'), 2-3 QCs and one water injection were included.Nicotiana attenuataNicotiana benthamianaNicotiana glaucaNicotiana tabacumLC raw files were processed by Refiner MS software from Genedata Expressionist® version 14.0.3 (http://www.genedata.com, accessed on 18 September 2022) (Giavalisco et al., 2011). The chromatography data processing output contained summed cluster abundances (“clustersum”), adding all detected isotopologue abundances. The abundance data were normalised to the abundance of the internal standard Crocin2 (CAS Number 55750-84-0, Sigma-Aldrich/Merck: PHL80392, manufacturer: Phytolab GmbH & Co.KG, Vestenbergsgreuth, Germany). The averaged background of the non-sample and water-only controls was subtracted from the initial abundances. Data were normalised to the amount of plant FW. The normalisation to the internal standard abundance levels accounts for potential variations due to matrix effects between samples and for manual handling errors during extraction. Crocin2 (also “Tricrocin” or “Crocetingentiobiosylglucosyl ester”) was chosen as an internal standard because it resembles the molecular properties of 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). It was the chemically closest of all commercially available standard compounds and had a terpenoid backbone of 20 carbon atoms as well. It harbours all-glucose glycosylations at both ends of the aglycone and resembles with its tri-glycosylation the average of the di- to penta-glycosylated HGL-DTGs.Specieshttps://www.ebi.ac.uk/metabolights/MTBLS13756ebert@mpimp-golm.mpg.deN. tabacum cv. Samsun NN (SNN) and N. glauca were grown together in one experiment (Exp 1), and a second batch of N. tabacum plants was grown together with N. benthamiana a second experiment (Exp 2, Figure S1). The plant lines of this study are long-term laboratory cultivars that are in-house propagated marker and reporter lines with either a hygromycine (N. tabacum) or a kanamycine resistance cassette and a YFP reporter gene (N. glauca and N. benthamiana) in their nuclear genomes (Stegemann and Bock, 2009; Stegemann et al., 2012; Fuentes et al., 2014). Plants were grown from seeds germinated on a synthetic medium and then transferred to soil (Exp 1) or initially raised in a sterile culture and then transferred to soil and grown to maturity (Exp 2) in a growth chamber in a 16 h light/8 h dark diurnal rhythm. The day conditions were 22°C, 75% humidity and a light intensity of 350 μmol photons m−2 s−1; the night conditions were 18 °C and 70% humidity. The aboveground biomass, with the exception of senescent or damaged leaves and the lower part of the stem, was harvested at a developmental stage when the plants had approximately 10 fully expanded leaves. The plant material was directly frozen in liquid nitrogen and stored until processing at −80°C. Samples were ground with mortar and pestle under liquid nitrogen. N. attenuata control plants were grown in tissue culture and directly frozen in liquid nitrogen at harvest.Metabolomicsultra-performance liquid chromatography-mass spectrometrytandem mass spectrometryNicotiana17-hydroxygeranyllinaloolnicotianoside IIcellular defense responseuntargeted metabolitesditerpene glycosidenicotianoside Itargeted metabolitesSecondary Metabolismultra-performance liquid chromatography-mass spectrometrytandem mass spectrometryNicotiana17-hydroxygeranyllinaloolnicotianoside IIcellular defense responseuntargeted metabolitesditerpene glycosidenicotianoside Itargeted metabolitesSecondary MetabolismFor detection, molecules were ionised by electrospray ionisation. Full mass range spectra were acquired in ESI(+) and ESI(-) ionisation modes ranging from mass to charge ratio (m/z) 100 to 1500 during the 0–19 min chromatography period. MS/MS spectra of metabolites were acquired in ESI(+) and ESI(-) mode at a collision energy of 25 eV by data-dependent tandem mass spectrometry (ddMS2). FT resolution was 25,000 for MS and 7500 for ddMS2. See (Giavalisco et al., 2011) for more detailed LC, MS and MS/MS settings.630.4 (Lyciumoside I)776.4 b (Lyciumoside IV)938.5 b (Attenoside)<h4>Background</h4>Specialised anti-herbivory metabolites are abundant in the solanaceous genus Nicotiana. These metabolites include the large family of 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). Many HGL-DTGs occur exclusively within the Nicotiana genus, but information from the molecular model species N. tabacum, N. benthamiana, and the tree tobacco N. glauca is limited.<h4>Objectives</h4>We studied HGL-DTG occurrence and complexity in these species with the aim of providing in-depth reference annotations and comprehensive HGL-DTG inventories.<h4>Methods</h4>We analysed polar metabolite extracts in comparison to the previously investigated wild reference species N. attenuata using positive ESI(+) and negative ESI(-) mode electrospray ionisation LC-MS and MS/MS.<h4>Results</h4>We provide annotations of 66 HGL-DTGs with in-source and MS/MS fragmentation spectra for selected HGL-DTGs with exemplary fragment interpretations of ESI(+) as well as less studied ESI(-) spectra. We assemble a potential biosynthesis pathway comparing the presence of HGL-DTGs in N. tabacum, N. glauca, and N. benthamiana to N. attenuata. Approximately one-third of HGL-DTGs are chromatographically resolved isomers of hexose, deoxyhexose, or malonate conjugates. The number of isomers is especially high for conjugates with low numbers of deoxyhexose moieties.<h4>Conclusions</h4>We extend the number of known HGL-DTGs with a focus on Nicotiana model species and demonstrate that the HGL-DTG family of N. tabacum plants can be surprisingly complex. Our study provides an improved basis with detailed references to previous studies of wild Nicotiana species and enables inference of HGL-DTG pathways with required enzymes for the biosynthesis of this important family of specialised defence metabolites.Detailed Profiling of 17-Hydroxygeranyllinalool Diterpene Glycosides from Nicotiana Species Reveals Complex Reaction Networks of Conjugation Isomers.Ebert Alina A, Alseekh Saleh S, D'Andrea Lucio L, Roessner Ute U, Bock Ralph R, Kopka Joachim JfalseDetailed Profiling of 17-Hydroxygeranyllinalool Diterpene Glycosides from Nicotiana Species Reveals Complex Reaction Networks of Conjugation IsomersBackground: Specialised anti-herbivory metabolites are abundant in the solanaceous genus Nicotiana. These metabolites include the large family of 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). Many HGL-DTGs occur exclusively within the Nicotiana genus, but information from the molecular model species N. tabacum, N. benthamiana, and the tree tobacco N. glauca is limited. Objectives: We studied HGL-DTG occurrence and complexity in these species with the aim of providing in-depth reference annotations and comprehensive HGL-DTG inventories. Methods: We analysed polar metabolite extracts in comparison to the previously investigated wild reference species N. attenuata using positive ESI(+) and negative ESI(-) mode electrospray ionisation LC-MS and MS/MS. Results: We provide annotations of 66 HGL-DTGs with in-source and MS/MS fragmentation spectra for selected HGL-DTGs with exemplary fragment interpretations of ESI(+) as well as less studied ESI(-) spectra. We assemble a potential biosynthesis pathway comparing the presence of HGL-DTGs in N. tabacum, N. glauca, and N. benthamiana to N. attenuata. Approximately one-third of HGL-DTGs are chromatographically resolved isomers of hexose, deoxyhexose, or malonate conjugates. The number of isomers is especially high for conjugates with low numbers of deoxyhexose moieties. Conclusions: We extend the number of known HGL-DTGs with a focus on Nicotiana model species and demonstrate that the HGL-DTG family of N. tabacum plants can be surprisingly complex. Our study provides an improved basis with detailed references to previous studies of wild Nicotiana species and enables inference of HGL-DTG pathways with required enzymes for the biosynthesis of this important family of specialised defence metabolites.2026-02-042026-01-23MTBLS13756MTBLC67236MTBLC67240MTBLC16721239452943CHEBI:67236CHEBI:67240CHEBI:167212