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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14210/m_MTBLS14210_LC-MS_positive_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14210/m_MTBLS14210_LC-MS___metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14210/a_MTBLS14210_LC-MS_positive_reverse-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14210/a_MTBLS14210_LC-MS___metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14210/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14210/s_MTBLS14210.txtprimaryOK200lower digestive tractftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14210Metabolite identification and annotation were performed using Thermo Scientific Compound Discoverer 3.3 software, in strict accordance with the Metabolomics Standards Initiative (MSI) guidelines for identification reliability classification. 1. Reference Databases and Spectral LibrariesMetabolite annotation was performed against authoritative, peer-reviewed databases and spectral libraries, covering primary metabolites, lipids and gut microbiota-related metabolites matched to mouse fecal samples:(1) MS/MS spectral libraries: mzCloud High-Resolution MS/MS Library (version 2024), NIST 2023 Tandem Mass Spectral Library, and an in-house validated metabolite standard spectral library.(2) Chemical and metabolite databases: Human Metabolome Database (HMDB, version 5.0), ChEBI (Chemical Entities of Biological Interest, release 226), LIPID MAPS (Lipid Metabolites and Pathways Strategy, version 2024), and PubChem Compound Database. 2. Annotation Matching Parameters(1) MS1 matching parameters: Monoisotopic mass tolerance ≤5 ppm, retention time tolerance ≤0.2 min, with matching of molecular formula and isotopic distribution pattern.(2) MS/MS matching parameters: Fragment ion mass tolerance ≤10 ppm, minimum fragment ion matching score of 70 (out of 100), with matching of normalized collision energy and fragmentation pattern.(3) Adduct ion matching: Common adducts were included for annotation, with [M+H]+, [M+Na]+, [M+NH4]+ for positive ionization mode, and [M-H]-, [M+Cl]-, [M+HCOO]- for negative ionization mode. 3. Identification Reliability ClassificationAll annotated metabolites were classified into the 4-level MSI identification system, which was clearly labeled in the submitted Metabolite Assignment File:(1) Level 1: Confidently identified metabolites, verified by matching both retention time and MS/MS spectrum with authentic chemical standards analyzed under identical chromatographic and mass spectrometric conditions.(2) Level 2: Putatively annotated metabolites, matched with MS/MS spectral libraries and public reference databases (the majority of annotated metabolites in this study), without standard verification.(3) Level 3: Putatively characterized compound classes, with confirmed chemical formula and compound category (e.g., fatty acids, glycerophospholipids) based on MS/MS fragmentation patterns, without specific structure annotation.(4) Level 4: Unknown features, detected and retained after quality filtering, without structural or chemical annotation. 4. Annotation CurationAll primary annotation results were manually curated to remove false positive matches, redundant annotations and misidentified metabolites. The final curated metabolite list, including chemical name, molecular formula, monoisotopic mass, m/z, retention time, database identifiers and identification reliability, was compiled into the Metabolite Assignment File for submission.mass spectrometry assayFecal metabolite extraction was performed on fecal samples from C57BL/6J mice with radiation-induced intestinal injury, including 4 experimental groups (Control, IR+PBS, IR+DY802, IR+TG-CS@DY802, n=3 per group). 1. Sample PreprocessingFecal samples were retrieved from a -80℃ ultra-low temperature freezer, and 30 mg of fecal pellets were accurately weighed into a sterile 2 mL EP tube on ice to avoid metabolite degradation. 2. Metabolite Extraction Procedure(1) 20 μL of internal standard (2-chloro-L-phenylalanine, 10 μg/mL) was added to each sample for quantitative correction and system stability monitoring.(2) 200 μL of pre-cooled ultrapure water was added, followed by 800 μL of pre-cooled methanol/acetonitrile mixture (1:1, v/v, LC-MS grade).(3) The samples were homogenized at 60 Hz for 2 min, then sonicated in an ice-water bath for 30 min to facilitate full metabolite release.(4) The samples were incubated at -20℃ for 1 h to precipitate proteins, followed by centrifugation at 14000 × g for 15 min at 4℃.(5) 800 μL of the supernatant was carefully transferred to a new sterile EP tube, and dried in a vacuum concentrator at room temperature without heating.(6) Before LC-MS analysis, the dried extract was reconstituted with 100 μL of acetonitrile/water mixture (1:1, v/v, LC-MS grade), vortexed for 1 min, sonicated in an ice-water bath for 10 min, and centrifuged at 14000 × g for 15 min at 4℃. The supernatant was transferred to a brown LC-MS injection vial for analysis. 3. Quality Control (QC) and Control Samples Preparation(1) Pooled QC sample: A pooled QC sample was prepared by mixing equal volumes of supernatant from all 12 experimental samples, and processed with the same extraction and reconstitution procedure as above. The QC sample was injected every 6 experimental samples throughout the analytical run to monitor the stability, repeatability and signal drift of the LC-MS system.(2) Solvent blank: A solvent blank sample (acetonitrile/water 1:1, v/v) was prepared and injected at the beginning and end of the run, as well as every 12 samples, to monitor and eliminate background contamination and carryover.(3) Procedure blank: A procedure blank sample without fecal matrix was processed with the same extraction procedure to exclude contamination from reagents and consumables. Mus musculusRaw LC-MS data processing and transformation were performed using Thermo Scientific Compound Discoverer 3.3 software, with a standardized workflow for untargeted metabolomics, including the following sequential steps: 1. Raw Data ImportRaw mass spectrometry data (.raw format) from both positive and negative electrospray ionization modes were directly imported into the software, with chromatographic and mass spectrometric parameters matched to the experimental acquisition method. 2. Molecular Feature Detection and Retention Time Alignment(1) Feature detection was performed with the following core parameters: mass tolerance of 5 ppm for full MS scan, minimum peak intensity threshold of 100000, minimum peak width of 0.05 min, maximum peak width of 2.0 min.(2) Retention time alignment was performed across all experimental samples, pooled QC samples and blank samples, with a maximum retention time shift tolerance of 0.2 min, to correct for systematic retention time drift during the analytical run. 3. Background Contamination Removal and Low-Quality Feature Filtering(1) Blank subtraction: Features detected in solvent blank and procedure blank samples were removed from the feature list, with a blank/sample intensity ratio threshold of 0.3, to eliminate background contamination and solvent interference.(2) De-isotoping: Isotopic peaks were removed to retain only the monoisotopic peak of each feature, avoiding redundant feature counting.(3) Low-quality feature filtering: Features with a detection rate <70% in all experimental samples and <80% in pooled QC samples were excluded, to ensure the reliability of retained features. 4. Quality Control Correction and Data Normalization(1) Signal drift correction: Locally estimated scatterplot smoothing (LOESS) regression-based signal correction was performed using pooled QC samples injected throughout the run, to correct for systematic signal intensity drift of the LC-MS system.(2) Internal standard normalization: Normalization was performed using the spiked internal standard (2-chloro-L-phenylalanine) to correct for injection volume variation and sample matrix effects.(3) Missing value imputation: Remaining missing values were imputed with 1/2 of the minimum positive intensity value in the dataset, to support subsequent statistical analysis. 5. Processed Data ExportThe final processed feature intensity matrix, including m/z, retention time and relative intensity of each feature in all samples, was exported for subsequent metabolite identification and statistical analysis.TG-CS@DY802 gavageDY802 gavagehttps://www.ebi.ac.uk/metabolights/MTBLS1421013612943895@163.comMice of C57BL/6 were received a single dose of 12 Gy of the whole abdominal radiation (WART) to establish acute radiation bowel injury model. Approximately 12 hours post-irradiation, mice were orally administrated with PBS, DY802 and TG-CS@DY802 respectively. For 5 days consecutive treatments, mice were suicided and collected with gut tissues.MetaboLightsPublicMetabolomicsLiquid Chromatography MS -Liquid Chromatography MS - positive - reverse-phaseThermo Scientific Vanquish UHPLC Systemlower digestive tractMus musculustargeted analysisMetabolic Processliquid chromatography mass spectrometry assayLipidomicsRadiation DamageThermo Scientific Q Exactive HFexperimental sampleChromatographic separation was performed on a Thermo Scientific Vanquish UHPLC System, with separate analytical methods for positive and negative electrospray ionization modes. 1. Chromatographic Column and Consumables(1) Analytical column: Waters ACQUITY UPLC BEH C18 Column (100 mm × 2.1 mm, 1.7 μm)(2) Guard column: Waters ACQUITY UPLC BEH C18 VanGuard Pre-column (5 mm × 2.1 mm, 1.7 μm)(3) Autosampler: Thermo Scientific Vanquish Autosampler 2. Chromatographic Parameters(1) Column temperature: 40℃(2) Autosampler temperature: 4℃(3) Injection volume: 2 μL(4) Flow rate: 0.3 mL/min(5) Needle wash: Needle was washed with 50% acetonitrile/water for 10 s after each injection to eliminate carryover. 3. Mobile Phase and Gradient Elution Program(1) Positive ionization mode:- Mobile phase A: 0.1% formic acid in ultrapure water (LC-MS grade)- Mobile phase B: 0.1% formic acid in acetonitrile (LC-MS grade)- Gradient elution program:0-1.0 min: 2% B1.0-9.0 min: 2% B to 98% B9.0-12.0 min: 98% B12.0-12.1 min: 98% B to 2% B12.1-15.0 min: 2% B (column equilibration) (2) Negative ionization mode:- Mobile phase A: 5 mM ammonium acetate in ultrapure water (LC-MS grade, pH 7.0)- Mobile phase B: Acetonitrile (LC-MS grade)- Gradient elution program:0-1.0 min: 2% B1.0-9.0 min: 2% B to 98% B9.0-12.0 min: 98% B12.0-12.1 min: 98% B to 2% B12.1-15.0 min: 2% B (column equilibration)Cell Host & Microbes.Thermo Scientific Vanquish UHPLC Systemlower digestive tractMus musculustargeted analysisMetabolic Processliquid chromatography mass spectrometry assayLipidomicsRadiation DamageThermo Scientific Q Exactive HFexperimental sampleNanfang Hospital, Southern Medical UniversityXinyi GuMass spectrometric detection was performed on a Thermo Scientific Q Exactive HF Orbitrap Mass Spectrometer, equipped with an electrospray ionization (ESI) source, operated in both positive and negative ionization modes with separate assay files for each mode. 1. General MS Parameters(1) Ion source: Heated electrospray ionization (HESI) source(2) Capillary temperature: 320℃(3) Aux gas heater temperature: 300℃(4) Sheath gas flow rate: 35 arb(5) Aux gas flow rate: 10 arb(6) S-lens RF level: 50(7) Mass scan range: 70-1050 m/z for both positive and negative modes(8) Orbitrap mass resolution: 120,000 (full MS scan), 30,000 (MS/MS scan) 2. Mode-Specific Parameters(1) Positive ionization mode: Spray voltage was set to 3.5 kV(2) Negative ionization mode: Spray voltage was set to 2.8 kV 3. Data Acquisition ModeData-dependent acquisition (DDA, Top 10) mode was used for MS and MS/MS data acquisition:(1) Full MS scan: AGC target 3e6, maximum injection time 100 ms(2) MS/MS scan: The top 10 most abundant precursor ions with intensity threshold >1e5 were selected for fragmentation by higher-energy collisional dissociation (HCD) with normalized collision energy (NCE) of 30 eV. AGC target was set to 1e5, maximum injection time was 50 ms, dynamic exclusion time was set to 15 s to avoid repeated fragmentation of the same precursor ion. 4. Mass CalibrationExternal mass calibration was performed before each analytical batch using Thermo Scientific Pierce LTQ Velos ESI Positive and Negative Ion Calibration Solutions, to ensure mass accuracy within 5 ppm for all detected ions.falseTargeted LC-MS analysis for lipidomics of mice intestinal samplesMice of C57BL/6 were received a single dose of 12 Gy of the whole abdominal radiation (WART) to establish acute radiation bowel injury model. Approximately 12 hours post-irradiation, mice were orally administrated with PBS, DY802 and TG-CS@DY802 respectively. For 5 days consecutive treatments, mice were suicided and collected with intestinal tissues for Liquid Chromatograph Mass Spectrometer (LC-MS) analysis of lipids and lipid-like molecules. 2026-04-022026-04-02MTBLS14210