MetaboLights

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Lftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab6_neg_Sample30.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab7_neg_sample21.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab4_POS_MS_SAMPLE-026.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/230404_Lab3_Sample_01_Pos.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab5_LTRS_03_Neg.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab1_IROA_RR_S17_neg.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab6_neg_Sample29.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab7_neg_sample13.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab6_pos_Sample03.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab6_pos_LTRS04.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab8_POS_Sample_31.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/230404_Lab3_Sample_10_Pos.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab7_pos_sample13.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab8_POS_Sample_17.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab5_sample28_Neg.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/LTRS_Lab8_POS_2.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab2_POS_Sample_4.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab1_IROA_RR_S26_neg.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab8_POS_Sample_04.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/230404_Lab3_Sample_05_Neg.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab8_NEG_Sample_09.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab7_pos_sample26.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab1_IROA_RR_S31_pos.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab7_pos_LTRS_02.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab6_neg_Sample16.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab5_sample19_Pos.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab1_IROA_RR_S15_pos.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab6_pos_Sample30.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab5_sample26_Pos.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab1_IROA_RR_S3_pos.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab4_NEG_MS_SAMPLE-019.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676/FILES/DERIVED_FILES/Lab6_neg_Sample03.mzXMLprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15676Metabolite annotations were assigned based on accurate mass and the m/z difference between the C12 and C13 peaks detected for a given feature. Molecular formulas were mapped to internal libraries in the ClusterFinder software to generate putative annotations.MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phaseLab 1LC system: Thermo Scientific Vanquish HPLCMobile Phase A: Water + 0.1% formic acidMobile Phase B: AcetonitrileColumn: Avantor ACE EXCEL 2 C18-PFP (2.1 x 100 mm, 2 um)Flow Rate: 0.35 mL/minInjection volume: 6 uL Lab 2LC system: Thermo Scientific Vanquish HPLCMobile Phase A: Water + 0.1% formic acidMobile Phase B: Methanol + 0.1% formic acidColumn: Acquity HSS T3 (2.1 x 150 mm, 1.8 um)Flow Rate: 0.3 mL/minInjection volume: 5 uL Lab 3LC system: Thermo Scientific Vanquish HPLCMobile Phase A: Water + 0.1% formic acid + 10 mM ammonium acetateMobile Phase B: Methanol + 0.1% formic acid + 10 mM ammonium acetateColumn: Phenomenex Kinetex C18 (2.1 x 150 mm, 2.6 um)Flow Rate: 0.2 mL/minInjection volume: 5 uL Lab 4LC system: SCIEX Exion UPLCMobile Phase A: Water + 0.1% formic acidMobile Phase B: Acetonitrile + 0.1% formic acidColumn: Phenomenex Luna Polar Omega (2.1 x 100 mm, 1.6 um)Flow Rate: 0.5 mL/minInjection volume: 10 uL Lab 5LC system: Agilent 1290 Infinity IIMobile Phase A: Water + 0.1% formic acidMobile Phase B: Acetonitrile + 0.1% formic acidColumn: Acquity HSS T3 (2.1 x 150 mm, 1.8 um)Flow Rate: 0.3 mL/minInjection volume: 5 uL Lab 6LC system: Waters nanoAcquity UPLCMobile Phase A: Water + 0.1% formic acidMobile Phase B: Acetonitrile + 0.1% formic acidColumn: Waters (2.1 x 150 mm, 1.8 um)Flow Rate: 0.07 mL/minInjection volume: 5 uL Lab 7LC system: SCIEX Exion LC 3.0Mobile Phase A: POS - Water + 0.2% formic acid | NEG – Water + 0.1% formic acid + 10 mM ammonium formateMobile Phase B: POS - Acetonitrile + 0.2% formic acid | NEG – AcetonitrileColumn: Phenomenex Luna Polar Omega (2.1 x 100 mm, 1.6 um)Flow Rate: 0.45 mL/minInjection volume: 10 uL Lab 8LC system: Agilent 1290 UPLCMobile Phase A: 95/5 Water/Acetonitrile + 0.1% formic acidMobile Phase B: 95/5 Acetonitrile/Water + 0.1% formic acidColumn: Waters Acquity CHS C18 (2.1 x 100 mm, 1.7 um)Flow Rate: 0.4 mL/minInjection volume: 5 uLMetabolomic profiles reproducibly differentiate clinically relevant, heterogeneous sample mixtures: a multi-lab “round robin” study.HALEY CHATELAINENational Center for Advancing Translational Sciences - National Institutes of Healthmixturemass spectrometry assayMeat homogenates were protein-normalized, and metabolites were extracted, centrally. Aliquots of extracts were distributed to all labs and maintained at -80 C until analysis.Gallus gallus; Sus scrofa; Bos taurusBos taurusSus scrofaGallus gallushttps://www.ebi.ac.uk/metabolights/MTBLS15676Haley Chatelaine. haley.chatelaine@nih.gov.Chris Beecher. chris@iroatech.com.Ewy Mathe. National Center for Advancing Translational Sciences. ewy.mathe@nih.gov.Data was transformed to mzXML using ProteoWizard. The mzXML files were input to ClusterFinder software for peak picking using the LTRS standards, which generated a library that was then applied for targeted peak picking on the samples. The picked and grouped peaks were output in a matrix as Clean Data. The Clean Data was then corrected for ion suppression to create the Suppression Corrected Data. Suppression correction involved dividing C12 peaks by the corresponding C13 standard peak and multiplying the resulting ratio by the mean integral of the C13 peak. The Suppression Corrected data was then normalized (SC Normalized Data) using a 'Dual MSTUS' (multiply each Suppression Corrected feature by the sum of the C12 peaks divided by the sum of the C13 peaks). A separate, normalized-only data set (Clean Normalized Data) by applying a MSTUS algorithm to the Clean Data.Relative concentrationSample grouphaley.chatelaine@nih.govSamples comprised homogenized, protein-normalized beef, chicken, and pork extracts. Seven sample solutions were prepared that comprised all possible equal mixtures of these three extract types: 1) Pure sample chicken (a), pork (b) or beef (c); 2) Dual mixtures of extracts (beef + pork (bc), beef + chicken (ac), pork + chicken (ab)); 3) Mix of all extracts (abc). Concentrations of pure of mixtures of extracts were varied as follows, resulting in 16 samples: Pure samples as 0.5, 1, and 1.5 concentration each (N=18); Dual mixtures at 0.3 and 0.5 concentration each (N=12); Mix of all extracts at 0.3 concentration each (N=2).Metabolomicspooled quality control sampleAgilent 6546 LC/Q-TOFuntargeted analysisZenoTOF 7600ring trial designisotope ratio mass spectrometryBos taurusAgilent 1290 Infinity UHPLCexperimental sampleThermo Scientific Vanquish UHPLC SystemThermo Scientific Q ExactiveThermo Scientific Orbitrap Fusion Lumos TribridSCIEX ExionLC ADWaters Xevo G2-XS QTofmixtureGallus gallus; Sus scrofa; Bos taurusShimadzu Nexera UHPLC systemuntargeted metabolitesSCIEX 5600 TripleToFWaters nanoACQUITY UPLC systemSus scrofaGallus galluspooled quality control sampleAgilent 6546 LC/Q-TOFuntargeted analysisZenoTOF 7600ring trial designisotope ratio mass spectrometryBos taurusAgilent 1290 Infinity UHPLCexperimental sampleThermo Scientific Vanquish UHPLC SystemThermo Scientific Q ExactiveThermo Scientific Orbitrap Fusion Lumos TribridSCIEX ExionLC ADWaters Xevo G2-XS QTofmixtureGallus gallus; Sus scrofa; Bos taurusShimadzu Nexera UHPLC systemuntargeted metabolitesSCIEX 5600 TripleToFWaters nanoACQUITY UPLC systemSus scrofaGallus gallusLab 1MS instrument: Thermo Scientific Q Exactive OrbitrapDrying gas temperature: 350Sheath gas temperature:Voltage: 3500 Vm/z range: 70-1000Scan rate: 6 scan/secondResolution: 35,000 Lab 2MS instrument: Thermo Scientific Orbitrap Fusion LumosDrying gas temperature:Sheath gas temperature: 350Voltage: 3500 Vm/z range: 70-1200Scan rate: 4 HzResolution: 120,000 Lab 3MS instrument: Thermo Scientific Orbitrap Fusion LumosDrying gas temperature: 250Sheath gas temperature:Voltage: 3500 Vm/z range: 70-1400Scan rate: 20 spectra/secondResolution: 240,000 Lab 4MS instrument: SCIEX 5600 Triple TOFDrying gas temperature: 400Sheath gas temperature: 40Voltage: 40 Vm/z range: 50-1000Scan rate: 8 scans/secondResolution: 30,000 Lab 5MS instrument: Agilent 6546 QTOFDrying gas temperature: 150Sheath gas temperature: 250Voltage: 2000 Vm/z range: 50-1700Scan rate: 5 spectra/secondResolution: Lab 6MS instrument: Waters Xevo G2-XS QTOFDrying gas temperature: 20Sheath gas temperature: 120Voltage: 3000 Vm/z range: 50-1200Scan rate: 2 spectra/secondResolution: Lab 7MS instrument: ZenoTOF 7600Drying gas temperature: 600Sheath gas temperature:Voltage: POS = 5500 V | NEG = 4500 Vm/z range: 50-1000Scan rate: Total scan time: 0.839 sec (0.1 s TOF + 25 SWATH win x 0.02s)Resolution: <0.02 Da Lab 8MS instrument: Agilent 6546 QTOFDrying gas temperature: 300Sheath gas temperature: 350Voltage: 3000 Vm/z range: 100-1700Scan rate: 1 spectrum/secondResolution:falseMetabolomic profiles generated using the IROA TruQuant platform result in reproducible differentiation of sample mixtures in a multi-lab “round robin” studyThe clinical utility of metabolomics hinges on reproducibly detecting differences between groups across analytical platforms. No multi-lab studies, to date, have attempted to evaluate the ability to deconvolve heterogeneous mixtures, which reflect mixtures of cell types present in clinical samples, using metabolomics profiles. We evaluated the reproducibility of the TruQuant metabolomics approach in eight labs in the US and UK. Each lab received identical aliquots of 32 samples, containing known mixtures of beef, pork, and chicken extracts of varying homogeneity levels. Metabolomic profiles were generated for each mixture in duplicate using each lab’s LC-MS methods. Anonymized, processed data were analyzed centrally. Despite differences in the number of features detected (560 – 1727 features), all labs correctly differentiated sample mixture types via unsupervised approaches. Pearson correlations of suppression corrected metabolite abundances, compared between lab pairs, were strong (mean = 0.87 ± 0.12 across all labs). Overall, this “round-robin” study design and data show that metabolomic analyses with appropriate study designs and controls enable reproducible conclusions across labs and platforms.2026-09-282026-09-15MTBLS15676