MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14029/m_MTBLS14029_LC-MS_alternating__metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14029/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14029/a_MTBLS14029_LC-MS_alternating__metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14029/s_MTBLS14029.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14029Target list of metabolites related to central carbon metabolism and nitrogen synthesis pathways including glycolysis, the TCA cycle, the urea cycle, peptidoglycan production, amino acids, and nucleoside synthesis was created using Agilent MassHunter Pathways to PCDL B.08.00 software. The software generates a target personal compound database library (PCDL) that contains names and chemical formulas. Addition of metabolite retention times in the target PCDL were provided from a pre-existing in-house database. This was then used for targeted features extraction in Agilent MassHunter Profinder 8.0 sp3.MetaboLightsPublicLiquid Chromatography MS - alternatingAqueous normal-phase liquid chromatography was performed using an Agilent 1290 Infinity II LC system equipped with a binary pump, temperature-controlled autosampler (set at 4°C) and temperature-controlled column compartment (set at 25°C) containing a Cogent Diamond Hydride Type C silica column (150 mm × 2.1 mm; dead volume of 315 µl). A flow rate of 0.4 mL/min was used. The elution of polar metabolites was performed using solvent A, which consisted of deionized water (resistivity ~18 MW cm) and 0.2% acetic acid, and solvent B, which consisted of 0.2% acetic acid in acetonitrile. The following gradient was used at a flow rate of 0.4 ml/min: 0 minute, 85% B; 0-2 minutes, 85% B; 3-5 minutes, 80% B; 6-7 minutes, 75% B; 8-9 minutes, 70% B; 10-11 minutes, 50% B; 11.1-14 minutes, 20% B; 14.1-25 minutes, 20% B; and 5-minute re-equilibration period at 85% B. Metabolomics profiling reveals changes in peptidoglycan and redox metabolism between ancient and modern Mycobacterium tuberculosis.Imperial College LondonYi LiuIntracellularmass spectrometry assayThe bacteria were metabolically quenched by plunging the filters into extraction solution composed of acetonitrile/methanol/H2O (2:2:1) precooled to 4°C. Small molecules were extracted by mechanical lysis of the entire bacteria-containing solution with 0.1 mm acid-washed zirconia beads for 1 minute using a FastPrep (MP Bio®) set to 6.0 m/second. The lysates were filtered twice through 0.22-μm Spin-X column filters (CoStar®). A 100μl aliquot of the metabolite solution was then mixed with 100 μl of acetonitrile with 0.2% acetic acid at -20°C and centrifuged for 10 minutes at 17,000 x g and 4°C. The final concentration of 70% acetonitrile was compatible with the starting conditions of chromatography. The supernatant was then transferred into LC-MS V-shaped vials for LC-MS analysis.Mycobacterium tuberculosishttps://www.ebi.ac.uk/metabolights/MTBLS14029Ashleigh Cheyne. Centre for Bacterial Resistance Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London. acheyne2@outlook.com.Gerald Larrouy-Maumus. Centre for Bacterial Resistance Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London. g.larrouy-maumus@imperial.ac.uk.Yi Liu. Centre of Bacterial Resistance Biology, Imperial College London. yi.liu13@imperial.ac.uk.The detected m/z data were deemed to be metabolites identified based on unique accurate mass-retention time and MS/MS fragmentation identifiers for masses exhibiting the expected distribution of accompanying isotopomers. The typical variation in abundance for most of the metabolites remained between 5 and 10% under these experimental conditions. Technical replicateBiological replicateyi.liu13@imperial.ac.ukThe Mtb strains were initially grown in 7H9 liquid medium containing the carbon sources of interest until the OD600 reached 0.8-1. 1 mL of bacteria was then loaded onto 0.22μm nitrocellulose filters under vacuum filtration. The mycobacteria-laden filters were then placed on top of chemically equivalent agar medium and allowed to grow at 37°C for 5 doubling times to generate sufficient biomass for targeted metabolomics studies.MetabolomicsMetabolomicstargeted metabolomic assaypeptidoglycanergothioneineMycobacterium tuberculosispeptidoglycanergothioneinetargeted metabolomic assayMetabolomicsMycobacterium tuberculosisAccurate mass spectrometry was performed using an Agilent Accurate Mass 6545 QTOF apparatus. Dynamic mass axis calibration was achieved by continuous infusion after chromatography of a reference mass solution using an isocratic pump connected to an ESI ionization source operated in the positive-ion mode. The nozzle and fragmentor voltages were set to 2,000 V and 100 V, respectively. The nebulizer pressure was set to 50 psig, and the nitrogen drying gas flow rate was set to 5 l/minute. The drying gas temperature was maintained at 300°C. The MS acquisition rate was 1.5 spectra/sec, and m/z data ranging from 50 to 1,200 were stored. This instrument enabled accurate mass spectral measurements with an error of less than 5 parts per million (ppm), a mass resolution ranging from 10,000 to 45,000 over the m/z range of 121-955 atomic mass units, and a 100,000-fold dynamic range with picomolar sensitivity. The data were collected in the centroid 4-GHz (extended dynamic range) mode. falseMetabolomics profiling reveals changes in peptidoglycan and redox metabolism between ancient and modern Mycobacterium tuberculosisMycobacterium tuberculosis (Mtb), the cause of tuberculosis, kills over one million people worldwide each year. Mtb is classified into ten distinct lineages, each harboring unique genetic changes that influence its survival, virulence, and transmissibility. Although metabolism plays a critical role in Mtb infection and persistence, the metabolomic profiles of different Mtb lineages remain poorly characterized. Here, by using metabolomic and bioinformatic approaches, we have determined the metabolome of representative strains belonging to three main lineages, namely lineage 1, lineage 2 and lineage 4. We show that the ancient lineage 1 has considerable differences in its metabolome compared to lineages 2 and 4. Those differences are mainly related to amino acids, peptidoglycan synthesis intermediates and ergothioneine, a sulphur-containing histidine derivative with potent antioxidant and redox properties. Taken together, these data represent the first comprehensive analysis of the metabolome of three main Mtb lineages.2026-06-262026-03-11MTBLS14029