MetaboLights

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showing a fold change of 1.5 or higher in the symbiosome space relative to each control (nodule cytosol or bacteroid lysate) were retained for analysis. Metabolites were annotated using KEGG IDs retrieved from the KEGG Medicago truncatula database (https://www.kegg.jp/kegg/pathway.html), and KEGG enrichment analysis was performed using TBtools.MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - hilicLiquid Chromatography MS - positive - reverse-phaseLiquid Chromatography MS - negative - hilicTwo complementary LC methods were used on a Vanquish UHPLC system (Thermo Fisher Scientific). Polar metabolites were separated on an ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 µm; Waters) with mobile phases of 25 mM ammonium acetate and 25 mM ammonium hydroxide in water (pH 9.75) (solvent A) and acetonitrile (solvent B). Non-polar metabolites were separated on a Kinetex C18 column (2.1 × 100 mm, 2.6 µm; Phenomenex) with mobile phases of 0.01% acetic acid in water (solvent A) and isopropanol:acetonitrile (1:1, v/v) (solvent B). The autosampler was maintained at 4 °C, and 2 µl of each sample was injected.Proteomic and metabolomic profiling depicts the functional landscape of the Medicago truncatula symbiosome.Institute of microbiology, CASJingxia Wuroot nodulemass spectrometry assayFor symbiosome space and nodule cytosol samples, 100 µl of each sample was extracted with 400 µl of methanol:acetonitrile (1:1, v/v) containing deuterated internal standards. Samples were vortexed for 30 s, sonicated for 10 min in a 4 °C water bath, and incubated at -40 °C for 1 h to precipitate proteins. For bacteroid fractions, 20 ± 1 mg of lyophilized sample was homogenized with beads in 1,000 µl of methanol:acetonitrile:water (2:2:1, v/v/v) containing deuterated internal standards, then subjected to three cycles of homogenization (35 Hz, 4 min) and sonication (5 min, 4 °C water bath), followed by incubation at -40 °C for 1 h to precipitate proteins. All samples were centrifuged at 13,800 g for 15 min at 4 °C, and supernatants were filtered under vacuum (6 psi, 120 s) prior to LC-MS/MS analysis. Quality-control (QC) samples were prepared by pooling equal aliquots of the supernatants from all samples.Medicago truncatulaSinorhizobium melilotihttps://www.ebi.ac.uk/metabolights/MTBLS15392Zhaosheng Kong. Institute of Microbiology, Chinese Academy of Sciences. zskong@im.ac.cn.Jingxia Wu. Institute of microbiology, CAS. wujingxia1996@gmail.com.Raw acquisition files were converted to mzXML format. Peak detection, alignment, and quantification were performed, and metabolite intensities were used for downstream statistical comparison across sample types.Sample typewujingxia1996@gmail.comSymbiosome space, bacteroid, and nodule cytosol fractions were isolated from root nodules of Medicago truncatula collected 4 weeks after inoculation with Sinorhizobium meliloti. Nodules were harvested, and symbiosomes were released and fractionated to obtain the symbiosome space fraction; bacteroids and nodule cytosol were collected in parallel as biological controls. Three biological replicates were prepared for each sample type.MetabolomicsbacteroidsMetabolomicsMedicago truncatulaquality controluntargeted analysisroot nodulesymbiosome spaceThermo Scientific Orbitrap Exploris 120nodule cytosolsymbiosomeThermo Scientific Vanquish UHPLC SystemXcaliburnitrogen fixationSinorhizobium melilotibacteroidsMedicago truncatulaMetabolomicsquality controlroot noduleuntargeted analysissymbiosome spaceThermo Scientific Orbitrap Exploris 120nodule cytosolsymbiosomeThermo Scientific Vanquish UHPLC SystemXcaliburnitrogen fixationSinorhizobium melilotiLC-MS/MS data were acquired on an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific) operating in information-dependent acquisition (IDA) mode controlled by Xcalibur software. Data were acquired in both positive and negative ionization modes for each chromatographic method (BEH Amide and C18).falseMetabolome of the Medicago truncatula symbiosome space, bacteroid, and nodule cytosolThis study characterizes the metabolome of the Medicago truncatula-Sinorhizobium meliloti symbiosis. Symbiosome space, bacteroid, and nodule cytosol fractions were isolated from 4-week-old root nodules and profiled by untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Polar metabolites were separated on a BEH Amide column and non-polar metabolites on a reversed-phase C18 column, each acquired in positive and negative ionization modes. Three biological replicates were analyzed per sample type, together with pooled quality-control samples. This dataset accompanies our proteomic and metabolomic profiling of the symbiosome and supports the finding that the symbiosome space is an active metabolic compartment enriched in soluble sugars and amino acids.2026-08-192026-08-19MTBLS15392