MetaboLights

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processing and analysis was performed with Matlab R2021b. Selection of peaks of metabolite i in j = N samples was as follows: 1) peaks with a prominence >0.1 were selected in the 12C and 13C channel of metabolite i; 2) If 12C and 13C maxima lay 5 data points next to each other, these peak pairs were retained; 3) The correlation of all peak pairs was determined using 11 data points around the maximum of the 12C peak, and the highest correlating peak pair was selected. After peaks were selected for sample 1 to N, the retention time occurring most often in all samples was determined as RTi = mode(RTij), where j = 1 − N. This ensured that the same peak pairs were selected for all samples. Subsequently the 12C peak and 13C peak next to RTi were selected in all samples and the correlation was again determined as described previously[1]. Ref:[1] Guder JC, Schramm T, Sander T, Link H. Time-Optimized Isotope Ratio LC-MS/MS for High-Throughput Quantification of Primary Metabolites. Anal Chem. 2017 Feb 7;89(3):1624-1631. doi:10.1021/acs.analchem.6b03731. PMID:28050903.MetaboLightsPublicLiquid Chromatography MS – HILICLiquid Chromatography MS - HILICSamples were analyzed by LC-MS/MS, with an Agilent 1290 Infinity II UHPLC system (Agilent Technologies) coupled to an Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies) as described previously[1]. Compound separation by liquid chromatography was achieved using 2 columns: i) an Acquity UPLC BEH Amide (Waters) for acidic conditions and ii) a iHILIC-Fusion(P) (HILICON AB) for basic conditions. The column oven was at 30 °C. LC solvents were: (A) water with ammonium formate (10 mM) and formic acid (0.1% v/v), (B) acetonitrile with formic acid (0.1% v/v) for acidic conditions; and (A) water with ammonium carbonate (10 mM) and ammonium hydroxide (0.2%), (B) acetonitrile for basic conditions. The LC gradient was: 0 min 90% B, 1.3 min 40% B, 1.5 min 40% B, 1.7 min 90% B, 2 min 90% B. The flow rate was 0.4 ml/min. The injection volume was 3 μl. Ref:[1] Guder JC, Schramm T, Sander T, Link H. Time-Optimized Isotope Ratio LC-MS/MS for High-Throughput Quantification of Primary Metabolites. Anal Chem. 2017 Feb 7;89(3):1624-1631. doi:10.1021/acs.analchem.6b03731. PMID:28050903.Homeostasis of the biosynthetic E. coli metabolome. 10.1016/j.isci.2022.104503. PMID:35754712University of TÃÂ¼bingenHannes LinkWhole Organismmass spectrometrySamples for metabolomics were collected at an OD600 of ca. 0.5, and 2 ml of the culture was vacuum-filtered through a filter membrane (HVLP02500, Merck Millipore). Subsequently, the filter was immersed into 1 ml of acetonitrile:methanol:H2O (40:40:20) at -20 °C. Extraction was performed overnight at -20 °C. Cell extracts were then centrifuged for 20 min at –9 °C, 13,000 rpm (Heraerus Pico 17 ThermoScientific). The supernatant of metabolite extracts was mixed with 13C internal standard in equal proportion and stored at -80 °C until analysis by LC-MS/MS. To prepare 13C internal standard, E. coli MG1655 was grown in a 1-L Bioreactor with 500 ml of M9 minimal medium containing 4 g/L uniformly 13C-labeled glucose (Cambridge Isotope Laboratories) to an OD of 1. The bioreactor was a BioFlo115 bioprocess system (Eppendorf, Hamburg, Germany), equipped with a pH-sensor (Mettler Toledo, Colombus, OH) and a DO-sensor (Mettler Toledo, Colombus, OH). The bioreactor was aerated using air passed through 4 M potassium hydroxide in order to avoid incorporation of 12C by CO2. Aliquots of 20 ml were vacuum-filtered using 0.45 μm pore size filters (HVLP02500, Merck Millipore) and immediately transferred into 5 ml of 40:40:20 (v-%) acetonitrile/methanol/water kept at -20 °C. Half of the culture was treated with a mixture of ampicillin, chloramphenicol, rifampicin, and trimethoprim for 15 min before sampling to enforces increases of metabolites. Final extracts from glucose-grown cells and inhibitor treated cells were mixed 1:1 and absence of 12C peaks in this 13C standard was confirmed by LC-MS. Aliquots of the 13C standard were stored at -80 °C. Alpha-ketoglutarate was derivatized prior to LC-MS/MS analysis[1], using 50 μM phenylhydrazine (Sigma-Aldrich). The mixture was incubated for 1 h at -20 °C in acetonitrile:methanol:H2O (40:40:20), and analyzed by LC-MS/MS on the Acquity UPLC BEH Amide column. Ref:[1] Zimmermann M, Sauer U, Zamboni N. Quantification and mass isotopomer profiling of α-keto acids in central carbon metabolism. Anal Chem. 2014 Mar 18;86(6):3232-7. doi:10.1021/ac500472c. PMID:24533614. Escherichia colihttps://www.ebi.ac.uk/metabolights/MTBLS4249Johanna Rapp. Interfaculty Institute for Microbiology and Infection Medicine Tübingen, University of Tübingen, Tübingen, Germany. johanna.rapp@uni-tuebingen.de.Hannes Link. Interfaculty Institute for Microbiology and Infection Medicine Tübingen, University of Tübingen, Tübingen, Germany. hannes.link@uni-tuebingen.de.Dušica Radoš. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.Stefano Donati. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.Martin Lempp.LC−MS/MS raw data (.d data files) were converted into .txt-files by using ProteoWizard MSconvert.ConditionCarbon source and other nutrientsGrowth rateReplicateMediumGrowth phase at samplingpHGrowth temperatureStrainhannes.link@uni-tuebingen.deBacterial strains:E. coli strains BW25113[1], MG1655 (DSMZ No. 18039) and NCM3722[2] were cultivated in M9 minimal medium in 19 experimental conditions. The cultivation conditions were the same as previously described[3]. Cultivation conditions:Glycerol-stocks of E. coli strains were kept at -80 °C and streaked out on LB plates (Luria Miller, Carl Roth), and incubated overnight at 37 °C. Single colonies were picked and inoculated into a M9 preculture (7.5 ml in 100-ml flasks), which was incubated for 6-10 h, to be subsequently diluted into a second preculture. The second preculture was used to inoculate the main culture at a starting OD of 0.05 in 35 ml medium in 500-ml non-baffled wide-neck shake flasks, covered by a 38-mm silicone sponge closure (BellCo glass). Cultivations were performed at 37 °C, 200 rpm and 5-cm shaking diameter (Infors HT Minitron), except for the heat-stressed cells which were grown at 42 °C. Growth was monitored by measuring the optical density at 600 nm (OD600). Specific growth rates were calculated by linear regression of ln(OD600) against the time during the exponential growth phase (see Table S1 in the paper associated with this study). Study design:For 17 conditions we used the E. coli strain BW25113, which was grown in: 1) minimal glucose medium, 2-11) minimal medium with 10 alternative carbon sources, 12-14) minimal glucose medium with 3 stresses (low pH of 6, high salt concentration, high temperature of 42 °C), 15-16) in stationary phase (1 d, 3 d), and 5), and 17) synthetic rich medium (minimal medium supplemented with glycerol, 20 amino acids, adenine and uracil). In another 2 conditions, we cultured the E. coli strains NCM3722 and MG1655 in minimal glucose medium. Biological samples were collected in triplicates from 200 ml shake flasks.Refs:[1] Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2:2006.0008. doi: 0.1038/msb4100050. PMID:16738554.[2] Soupene E, van Heeswijk WC, Plumbridge J, Stewart V, Bertenthal D, Lee H, Prasad G, Paliy O, Charernnoppakul P, Kustu S. Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression. J Bacteriol. 2003 Sep;185(18):5611-26. doi:10.1128/JB.185.18.5611-5626.2003. PMID:12949114.[3] Schmidt A, Kochanowski K, Vedelaar S, Ahrné E, Volkmer B, Callipo L, Knoops K, Bauer M, Aebersold R, Heinemann M. The quantitative and condition-dependent Escherichia coli proteome. Nat Biotechnol. 2016 Jan;34(1):104-10. doi:10.1038/nbt.3418. PMID:26641532.Metabolomicsultra-performance liquid chromatography-mass spectrometrytandem mass spectrometryisotope ratio mass spectrometrytargeted metabolitesHomeostatic Processultra-performance liquid chromatography-mass spectrometrytandem mass spectrometryisotope ratio mass spectrometrytargeted metabolitesHomeostatic ProcessEluting compounds were detected using a Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies) equipped with an Agilent Jet Stream electrospray ion source in positive and negative ion mode. Source gas temperature was set to 200 °C, with 14 L/min drying gas and a nebulizer pressure of 24 psi. Sheath gas temperature was set to 300 °C and flow to 11 L/min. The electrospray nozzle and capillary voltages were set to 500 and 2500 V, respectively. Compounds were selected in 12C- and 13C isoforms as described previously[1]. Ref:[1] Guder JC, Schramm T, Sander T, Link H. Time-Optimized Isotope Ratio LC-MS/MS for High-Throughput Quantification of Primary Metabolites. Anal Chem. 2017 Feb 7;89(3):1624-1631. doi:10.1021/acs.analchem.6b03731. PMID:28050903.UDP-N-acetyl-D-GlucosamineL-PhenylalanineRiboflavinUDP-GlucoseDihydropteroateOrotateHistidinolL-OrnithineN-Acetyl-D-Glucosamine-PL-HomoserineL-AlanineCytosineFlavin adenine dinucleotideL-HistidineIsopentenyl diphosphate / Dimethylallyl diphosphateSemicarbazidePentose-PSuccinateN1-(5-Phospho-D-ribosyl)glycinamide5-Phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazoleSeduheptulose 7-PL-ProlineL-Methionine5-Phospho-alpha-D-ribose 1-Diphosphate (PRPP)MalateHypoxanthineL-Aspartic acidL-LysineUrea (Carbamide)D-Alanyl-Alaninea-Ketoglutaric acidXanthosine 5-P3-HydroxypropanoateL-ArginineUDP-N-acetylmuramoyl-L-alanineN-Succinyl-L-2-amino-6-oxopimelate5-MethylthioadenosineIndolegamma-L-Glutamyl-L-cysteineGlycine(2R,3R)-2,3-Dihydroxy-3-methylpentanoateFolateGuanineL-AsparagineL-Leucine/L-IsoleucinedTDPD-Glucosamine-PL-TryptophanDihydroorotateDeoxyribose-P5-Phosphoribosyl-N-formylglycinamidinePyridoxamineShikimateReduced glutathioneL-Glutamic acidAdenosineDiaminopimelic acidN-Acetyl-L-Glutamic acidN-AcetylputrescineXanthosineErythrose 4-PAdenineN-Carbamoyl-L-aspartate5-Amino-1-(5-phospho-D-ribosyl)imidazoleGlycolaldehydedCMP1,5-Diaminopentanegamma-Butyrobetaine4-Methyl-2-oxovaleric acid (Ketoleucine)2-Dehydro-3-deoxy-D-arabino-heptonate 7-phosphate7,8-DiaminononanoateL-GlutamineAnthranilateL-SerineInosineD-Pantothenic aciddUMPN2-Succinyl-L-glutamateMetabolite concentrations vary across conditions and such metabolome changes are relevant for metabolic and gene regulation. Here, we used LC-MS/MS to explore metabolite concentration changes in Escherichia coli. We measured 101 primary metabolites in 19 experimental conditions that include various nutrients and stresses. Many metabolites showed little variation across conditions and only few metabolites correlated with the growth rate. The least varying metabolites were nucleotides (e.g. UTP had 10% variation) and amino acids (e.g. methionine had 13% variation). These results show that E. coli maintains protein and RNA building blocks within narrow concentration ranges, thus indicating that many feedback mechanisms in biosynthetic pathways contribute to end-product homeostasis.Homeostasis of the biosynthetic E. coli metabolome.Radoš Dušica D, Donati Stefano S, Lempp Martin M, Rapp Johanna J, Link Hannes HfalseHomeostasis of the biosynthetic E. coli metabolomeMetabolite concentrations vary across conditions and such metabolome changes are relevant for metabolic and gene regulation. Here, we used LC-MS/MS to explore metabolite concentration changes in Escherichia coli. We measured 101 primary metabolites in 19 experimental conditions that include various nutrients and stresses. Many metabolites showed little variation across conditions and only few metabolites correlated with the growth rate. The least varying metabolites were nucleotides (e.g. UTP had 10% variation) and amino acids (e.g. methionine had 13% variation). These results show that E. coli maintains protein and RNA building blocks within narrow concentration ranges, thus indicating that many feedback mechanisms in biosynthetic pathways contribute to end-product homeostasis.2022-11-022022-02-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:15380CHEBI:15351CHEBI:16474CHEBI:18009CHEBI:73200CHEBI:16908CHEBI:15846CHEBI:17659CHEBI:37736CHEBI:48000CHEBI:18406CHEBI:15763CHEBI:181907CHEBI:17239CHEBI:37515CHEBI:17052CHEBI:17455CHEBI:30769CHEBI:15999CHEBI:4167CHEBI:35128CHEBI:15978CHEBI:16108CHEBI:44897CHEBI:15350CHEBI:30763CHEBI:15346CHEBI:167606CHEBI:15996CHEBI:15422CHEBI:15713CHEBI:17677CHEBI:17552CHEBI:16761CHEBI:17345CHEBI:17202CHEBI:16027CHEBI:17489CHEBI:16695CHEBI:17361CHEBI:15682CHEBI:16750CHEBI:17705CHEBI:17562CHEBI:17895CHEBI:16349CHEBI:16414CHEBI:16865CHEBI:15539CHEBI:26355CHEBI:15919CHEBI:15641CHEBI:15633CHEBI:17808CHEBI:15414CHEBI:16680CHEBI:18337CHEBI:17001CHEBI:17256CHEBI:18335CHEBI:16160CHEBI:15811CHEBI:1467CHEBI:181895CHEBI:17275CHEBI:17712CHEBI:17981CHEBI:17568CHEBI:84726CHEBI:16264CHEBI:18066CHEBI:18319CHEBI:18075CHEBI:17111CHEBI:18413CHEBI:17622CHEBI:15784CHEBI:15721CHEBI:18150CHEBI:16584CHEBI:17363CHEBI:11563CHEBI:48153CHEBI:15859CHEBI:16119CHEBI:17025CHEBI:16742CHEBI:27512CHEBI:30915CHEBI:30797CHEBI:17053CHEBI:48430CHEBI:15741CHEBI:17071CHEBI:27470CHEBI:17596CHEBI:16335CHEBI:16828CHEBI:16026CHEBI:16467CHEBI:17295CHEBI:15971CHEBI:16235CHEBI:16643CHEBI:16015CHEBI:18019CHEBI:18050CHEBI:16255CHEBI:16708CHEBI:17196CHEBI:15729CHEBI:17191CHEBI:15699CHEBI:16881CHEBI:17203CHEBI:16040CHEBI:17115CHEBI:16977CHEBI:15428CHEBI:16199CHEBI:16238CHEBI:17015CHEBI:15652CHEBI:4581CHEBI:15918CHEBI:16856CHEBI:17509CHEBI:138560CHEBI:35266CHEBI:18349CHEBI:18107CHEBI:15873CHEBI:17515CHEBI:48957CHEBI:46905CHEBI:17533CHEBI:2247CHEBI:16410CHEBI:16576CHEBI:1941CHEBI:30754CHEBI:17368CHEBI:17768CHEBI:18127CHEBI:33404CHEBI:28306