MetaboLights

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To do this, the theoretical molecular formulas were entered as the “Target Source”. Additional settings were: Match tolerance: 5 ppm; ion species: +H, +Na, +K, -H2O+H; isotope model: common organic molecule; charge state: 1-2.MetaboLightsPublicLiquid Chromatography MS - positive - reverse-phaseChromatographic separation was achieved on a 1290 Infinity II HPLC (Agilent Technologies) equipped with a Poroshell 120 EC-C8 column (3.0 × 150 mm, 2.7 μm; Agilent Technologies) guarded by a precolumn (3.0 × 5 mm, 2.7 μm) of identical material. The column compartment was maintained at 50°C and the injection volume was 10 μl. A mobile phase system consisting of water/methanol (8:2, v:v; solvent A) and 2-propanol/methanol (9:1, v:v; solvent B), both acidified with 0.1% formic acid and 10 mM ammonium formate, was used for gradient elution at an initial composition of 45:55 (A:B, v:v) and a flow rate of 0.25 ml/min.Sphingolipids associate with the chlamydial nucleoid and mark developmental transitions in Chlamydia trachomatis.Chlamydia trachomatis deploys sphingolipids for genome organisation. 10.64898/2026.04.29.721357.Freie Universität Berlin, Institute of PharmacyFabian SchumacherHeLa cellmass spectrometry assayCell suspensions were subjected to lipid extraction using 1.5 ml methanol/chloroform (2:1, v:v) containing 17:0 ceramide (C17 Cer) and d31-16:0 sphingomyelin (d31-C16 SM) as internal standards. Extraction was facilitated by incubation at 48°C with gentle shaking (120 rpm) overnight. To reduce interference from glycerolipids, samples were saponified with 150 μl 1 M methanolic KOH for 2 h at 37°C with gentle shaking (120 rpm) followed by neutralization with 12 μl glacial acetic acid. After centrifugation at 2,200 g for 10 min at 4°C, organic supernatants were evaporated to dryness using a Savant SpeedVac concentrator (Thermo Fisher Scientific). Dried residues were reconstituted in 200 μl acetonitrile/methanol/water (47.5:47.5:5 (v:v:v), 0.1% formic acid) and subjected to QTOF-MS analysis.Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS15303Fabian Schumacher. Freie Universität Berlin, Institute of Pharmacy. Königin-Luise-Str. 2+4, 14195 Berlin, Germany. fabian.schumacher@fu-berlin.de.Agilent MassHunter Qualitative Software 10.0 was used to extract m/z values and to obtain LC-MS chromatograms and mass spectra.InfectionTreatmentReplicateIncubation timefabian.schumacher@fu-berlin.deHeLa 229 cells were seeded a day prior to the experiment in a 6-well plate (24 h time point: 5 × 10^5 cells per well for infected samples and 2.5 × 10^5 cells per well for uninfected samples, 40 h time point: 2.5 × 10^5 cells per well for infected samples and 1.25 × 10^5 cells per well for uninfected samples). The next day, the medium of the cells was exchanged with fresh standard medium. The cells were infected with C. trachomatis at MOI 1 or left uninfected. 3 h after infection, the medium was exchanged to medium containing 1 % heat-inactivated FBS and 10 μM TFSM1 for one well per time point and condition. At the indicated time points, cells were washed 2 × with DPBS, resuspended in 1 ml methanol.Metabolomicshigh-resolution mass spectrometryMassHunter Data Acquisitiontriple quadrupole instrument6495C Triple Quadrupole LC/MSHomo sapiensAccurate mass mode settingLipidomicsChlamydia trachomatisSphingolipidstargeted metabolite profilinglipid extractionsemi-targeted analysisMassHunter Qualitative Analysisexperimental sampleHigh Performance Liquid ChromatographyAgilent 6550 iFunnel Q-TOFQTOF-MStandem mass spectrometryinfectious diseaseHeLa cell6550 iFunnel Q-TOF LC/MSAgilent 1290 Infinity II UHPLCBioSupraMolMassHunter Quantitative Analysishigh-resolution mass spectrometryMassHunter Data Acquisitiontriple quadrupole instrument6495C Triple Quadrupole LC/MSAccurate mass mode settingLipidomicsHomo sapiensChlamydia trachomatisSphingolipidstargeted metabolite profilinglipid extractionsemi-targeted analysisMassHunter Qualitative AnalysisHigh Performance Liquid Chromatographyexperimental sampleQTOF-MSAgilent 6550 iFunnel Q-TOFtandem mass spectrometryinfectious diseaseHeLa cell6550 iFunnel Q-TOF LC/MSAgilent 1290 Infinity II UHPLCBioSupraMolMassHunter Quantitative AnalysisMetabolite detection was performed after positive electrospray ionization (ESI+) in full-scan mode, in the m/z range of 50–1000 and at a scan rate of 3 spectra/s.falseIdentification of metabolites of trifunctional sphingomyelin (TFSM1) in HeLa 229 cells infected with Chlamydia trachomatisChlamydia trachomatis is an obligate intracellular bacterial pathogen and a leading cause of sexually transmitted infections worldwide. During its biphasic developmental cycle, infectious, non-replicative elementary bodies alternate with replicative reticulate bodies within a membrane-bound intracellular niche known as the inclusion. C. trachomatis relies heavily on host-derived metabolites, including sphingolipids, which are essential for inclusion integrity, bacterial growth and production of infectious progeny. Here, using expansion microscopy, we uncover an unexpected localization of sphingolipid derivatives within the highly condensed DNA nucleoids of elementary bodies. These sphingolipids are released from nucleoids prior to DNA decondensation during the elementary-to-reticulate body transition, the earliest phenotypic event in the complex developmental cycle of these bacteria. Thereafter, nucleoids undergo a characteristic DNA decondensation process that we visualized by expansion microscopy. By combining super-resolution imaging with a FRET-based metabolic tracking approach and lipidomics, we identified sphingomyelin derived from the sphingolipid analogues as the sphingolipid species predominantly associated with the condensing nucleoids of elementary bodies. Notably, reticulate bodies arrested in their developmental stage fail to accumulate sphingomyelin, suggesting a potential role for this lipid in stage-specific DNA condensation. The study includes high-resolution QTOF-MS data for the identification of metabolites of functionalized sphingomyelin (TFSM1) in HeLa cells infected with C. trachomatis.2026-08-102026-08-10MTBLS15303