MetaboLights

application/xml

ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14002/m_MTBLS14002_LC-MS_negative__metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14002/m_MTBLS14002_LC-MS_positive__metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14002/a_MTBLS14002_LC-MS_negative__metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14002/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14002/a_MTBLS14002_LC-MS_positive__metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14002/s_MTBLS14002.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14002Lipid annotation was performed using Bruker MetaboScape 2025b by combining accurate mass, isotope pattern, retention time, ion mobility (CCS), and MS/MS spectral matching. Lipid annotation was carried out using the Lipid Species Annotation module and lipid-focused MS/MS spectral libraries, including MoNA LipidBlast 2022 and MoNA HMDB. Internal standards from the Avanti SPLASH Lipidomix mixture were annotated using target lists containing expected formulas, retention times, and CCS values.Annotations were accepted based on predefined thresholds for m/z error, mSigma, CCS agreement, and MS/MS score, followed by manual review of diagnostic fragment ions and spectral quality. For features lacking confident MS/MS-based annotation, accurate-mass matching was performed using additional orthogonal constraints, including expected retention behavior, Kendrick mass defect patterns, expected adduct forms, ion mobility consistency, and biochemical plausibility for the sample matrix. Final annotations were reported as putative lipid species-level assignments unless confirmed with authentic standards.MetaboLightsPublicLiquid Chromatography MS - positiveLiquid Chromatography MS - negativeUntargeted lipidomic analysis was performed using a Bruker timsTOF Pro mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a VIP-HESI source and coupled to an Agilent 1290 Infinity III UHPLC system (Agilent Technologies, Santa Clara, CA, USA). Lipid separation was achieved using an ACQUITY UPLC CSH C18 column (2.1 × 100 mm, 1.7 µm; Waters Corporation, Milford, MA, USA) maintained at 50 °C.Mobile phase A consisted of 10 mM ammonium formate in methanol/acetonitrile/water (40:35:25, v/v/v) and mobile phase B consisted of 10 mM ammonium formate in 2-propanol/acetonitrile/water (96:3:1, v/v/v). The flow rate was 0.35 mL/min, with gradient elution starting at 10% MPB, increasing to 99% MPB, and returning to 10% MPB over a 20 min run.Lipid extracts were resuspended in 1:1 mobile phase A/mobile phase B prior to analysis. Injection volumes were 5 µL for positive ionization mode and 10 µL for negative ionization mode. The autosampler was maintained at 4–8 °C.Untargeted lipidomics of mammalian serum using LC-TIMS-PASEF.University of CalgaryFernanda Sousa Monteiroblood serummass spectrometry assaySerum lipids were extracted using a miniaturized liquid–liquid extraction based on the Folch method optimized for untargeted LC–MS lipidomics. For each extraction batch, 10 samples, one pooled quality control (QC) sample, and one extraction blank were processed in parallel. The extraction blank consisted of water processed identically to biological samples to monitor background contamination.Frozen serum aliquots stored at −80 °C were kept on ice during processing. Prior to extraction, 4 µL of the Avanti SPLASH Lipidomix internal standard mixture (Avanti Research) was added to each sample, QC, and blank to enable signal normalization and monitoring of extraction performance. Methanol was then added to induce protein precipitation, followed by dichloromethane and an aqueous 0.2 M potassium chloride solution to generate a biphasic system (dichloromethane:methanol:aqueous ratio of 8:4:3). Samples were vortexed, incubated at 4 °C, and centrifuged to achieve phase separation.Following centrifugation, the lower organic phase containing extracted lipids was carefully collected and transferred to clean tubes. Organic extracts were evaporated to dryness using nitrogen blow-down or vacuum centrifugation and stored at −80 °C until LC–MS analysis. All extractions were performed in a randomized order to minimize batch effects and potential systematic bias. Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS14002Fernanda Sousa Monteiro. University of Calgary. fernanda.sousamontei@ucalgary.ca.Adriana Zardini Buzatto. University of Calgary. adriana.zardinibuzat@ucalgary.ca.Raw LC–MS/MS data were processed separately for positive and negative ionization modes using Bruker MetaboScape 2025b. Data transformation included mass recalibration using sodium formate, ion mobility recalibration using tuning mix reference values, lock-mass correction, feature detection, retention time alignment, and MS/MS import within the chromatographic window excluding the calibration segment. Feature extraction was performed using the T-ReX 4D algorithm with intensity, peak-size, occurrence, and group-presence filters optimized for untargeted lipidomics.Following vendor processing, curated feature tables were exported for downstream processing in Excel and custom Python scripts. Additional transformation steps included removal of blank-associated signals, low-intensity and artifactual features, filtering based on pooled QC reproducibility, missing value imputation using low-intensity dataset-derived values, internal-standard normalization, and QC-based drift correction when required. Positive- and negative-ionization datasets were processed independently prior to final merging for downstream statistical analysis.Replicatefernanda.sousamontei@ucalgary.caHuman serum samples used in this study consisted of commercially available pooled normal human serum (MilliporeSigma, Cat. No. S1-100ML). The material was obtained as a ready-to-use biological matrix derived from healthy human donors and supplied by the manufacturer under standardized collection and processing conditions. Upon receipt, serum aliquots were prepared to minimize repeated freeze–thaw cycles and stored at −80 °C until analysis. Prior to lipid extraction and LC–MS analysis, aliquots were thawed on ice and gently mixed to ensure sample homogeneity. No additional biological treatments or time-course exposures were applied to the serum samples prior to analysis.Metabolomicsliquid chromatography mass spectrometry assayblood serumLipidomicslipid extractionliquid chromatography mass spectrometry assayblood serumLipidomicslipid extractionMass spectrometric detection was performed using a timsTOF Pro mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a VIP-HESI electrospray ionization source. Data were acquired in both positive and negative ionization modes using trapped ion mobility spectrometry–parallel accumulation serial fragmentation (TIMS-PASEF).Mass spectra were acquired over the m/z range 120–1850, with two PASEF ramps per acquisition cycle and a total cycle time of 0.36 s. Ion mobility separation was performed over a 1/K0 range of 0.60–2.00 V·s/cm², with ramp and accumulation times of 65 ms.Source parameters included a capillary voltage of 4300 V, an end plate offset of 500 V, a nebulizer pressure of 2.0 bar, a dry gas flow of 8 L/min, and a dry gas temperature of 220 °C. The sheath gas temperature was 300 °C with a sheath gas flow of 3 L/min.Fragmentation was performed using collision-induced dissociation (CID) with mobility-dependent collision energies ranging from 22–37 eV in positive mode and 21–39 eV in negative mode. Mass and ion mobility calibration were performed prior to analysis using sodium formate and ESI tuning mix standards.Cer 44:1;O4Cer 44:1;O3PC 18:1_20:3PC 18:1_20:4Cer 44:1;O2SHexCer 40:1;O2HexCer 34:0;O3HexCer 34:0;O2LPC 20:2;OHexCer 38:1;O2PC O-32:1HexCer 38:1;O3PC O-32:3FA 19:0PC O-32:2BMP 32:0PC O-32:5HexCer 32:5;O2LPE 24:1FA 19:2PC O-32:4LPE 24:2FA 19:1PG O-32:4LPE 24:3LPE O-14:0PC O-32:6PE 38:5PG O-32:2FA 20:4;O5PE 38:4PG 36:3;OPG O-32:3PE 38:3SM 40:3;O2LPC 18:5;OFA 20:4;O2FA 20:4;O3FA 20:4;O4PC 36:3;OCer 36:2;O4FA 18:2;OPC O-44:7Cer 36:3;O2PC O-44:8Cer 36:3;O4Cer 40:0;O3PS 44:2;OCer 40:0;O4PI 16:0_18:2PC 18:0_24:4PC 30:2FA 20:3;O4PC 30:3PG 16:0_18:2FA 20:3;O5PC 30:0PE 16:0_18:2;OHex-NeuAc-Cer 38:1;O2FA 20:3;O2FA 20:3;O3PC 34:6;OPI 34:0;OST 26:2;O2HexCer 44:5;O3PC 15:0_16:0Cer 32:1;O2PE 46:5;OFA 18:2;O4PI 18:0_20:2FA 18:1PI 18:0_20:4FA 18:0PC O-33:4FA 18:3PC O-33:3FA 18:2HexCer 40:3;O2FA 18:2;O3FA 18:4PG 36:2;OPE 37:3PE 18:0_22:4PC 42:6LPI 17:0;OPE 18:0_22:5PC 36:2;OPE 37:1FA 18:3;OPS 30:1;OPI 38:4PI 38:2Hex2Cer 42:0;O3PE 16:0_18:0PMeOH 16:0_18:1PE 16:0_18:1ST 26:4;O2;GlyCer 36:1;O4PC 34:5;OCer 36:1;O3PE 16:0_18:3Cer 36:1;O2PE 38:4;OLPE 22:0LPE 22:1LPE 22:2PE O-38:5PG 32:2FA 20:1;OPS 28:0PE O-38:7PC O-30:3BMP 34:1PC O-30:2LPE 24:3;OPC 40:0LPE 22:4FA 18:0;O2PC 41:6Cer 44:2;O4PC O-30:0PI 40:5;OSM 34:0;O2PC 36:1;OLPA 16:3Cer 32:0;O2SHexCer 36:4;O3HexCer 38:0;O2PE 48:3;OSPB 20:3;OLPC 20:3PC 34:4;OCer 44:3;O4PG 16:0_16:1LPC 20:2Hex2Cer 44:4;O3PMeOH 18:0_20:4PG 33:1PC O-31:4PE 37:5;OPC O-31:3SM 38:1;O2FA 26:2;O2FAHFA 32:3;OFA 26:2;O3PC 15:0_18:2PC 40:3FA 18:1;OFA 20:2;O2PC 40:4PG 36:4;OPC 40:5PC 40:6ST 26:1;O2NeuAcHexCer 34:1;O2PE 18:0_20:2PE 18:0_20:1PI 36:5PE 18:0_20:3SPB 20:2;OLPE O-22:2PI 36:2FA 18:1;O3Cer 18:1;2O/18:0ST 24:5;O3NAE 18:3;OFA 20:1;O3PE 16:0_16:1PC 34:3;OFA 20:3;OPC O-36:5LPE 20:1PC O-36:4PC 18:2_20:0PC O-36:6LPE 20:4LPE O-18:1LPE O-18:2LPE 20:0LPC 18:3PC O-36:1NAT 18:1PC 18:2_20:1FA 18:4;O2PC O-36:3PC O-36:2PS 34:3;OPG 18:1_18:1;OLPC 18:0LPC 18:1PC 18:0_20:3LPC 18:1;OPG 16:1_18:1HexCer 42:1;O3PE 16:0_20:4;OPS 30:4PS 30:3Cer 38:0;O3PE O-40:6Cer 38:0;O4PE O-40:8PS 42:3PE 42:4;OPS 42:5PC 34:2;OST 21:1;O3LPA 17:0PC O-37:4PA 35:2HexCer 42:2;O3FA 20:2;OSHexCer 40:3;O3PE O-22:1_22:6PC 16:0_16:1PMeOH 16:0_20:4Cer 38:1;O3Cer 20:2;O2/16:0Cer 38:1;O2Cer 38:1;O4PC O-37:1PS 34:2;OPE 30:3;O3LPC 19:0LPC 18:0;OCer 44:4;O4PC 32:1;OFA 18:5;O2Hex2Cer 40:1;O2HexCer 32:2;O2Hex2Cer 40:1;O3HexCer 32:2;O3NAE 22:5;OLPC O-22:6LPS 26:1;OCer 42:5;OFA 20:0SHexCer 38:5;O3PS 28:0;OSM 40:4;O2FA 20:1FA 20:4PE 17:0_18:1FA 20:3FA 20:5PE 40:3PC 34:1;OFA 20:5;OPE 40:6LPE O-16:0PC O-34:6PC 33:3;ONAE 16:1;OPC O-34:1PC O-34:0PC O-34:3PC O-34:2PC O-34:4PC 32:2;OPE 20:0_20:4PA 38:4;OHexCer 34:1;O3PS 18:0_18:2PC 18:1_18:2PS 18:0_18:1SHexCer 40:2;O2FA 22:0;OPC O-46:3FA 18:3;O2FA 18:3;O3HexCer 32:4;O2PS 40:1HexCer 30:5;O2PS 40:3PS 40:4PS 40:5PS 40:6PG 18:0_18:1FA 20:4;OFA 16:1;OPC O-35:6PC O-35:5PA 37:2PC 16:0_18:0Hex2Cer 40:0;O3PC O-35:4PC 16:0_18:2PS 40:0HexCer 32:3;O2SM 40:5;O2NAE 22:2;O2LPA 19:1LPC O-20:2LPC O-20:0HexCer 42:3;O2HexCer 42:3;O3Cer 38:2;O2Cer 38:2;O4NAE 22:3;OFA 22:0FA 22:2FA 22:1LPC 16:1;OFA 22:3PE 28:2;O3PE 42:4FA 22:4;O4PG 38:3PG 38:2PE O-32:5LPC 14:0PC 32:4;OPI 40:5PI 40:4PE O-32:1FA 16:1;O2PE 18:0_18:2;OCer 42:1;O4PG 16:0_22:6FA 22:4;O2PS 34:3CerP 44:2;O2Hex-NeuAc-Cer 36:1;O2PE O-44:4PS 40:2;OPE O-42:2FA 21:0CAR 22:6Cer 42:0;OPC 38:0Cer 42:0;O3PC 38:1HexCer 36:1;O3Cer 42:0;O4HexCer 36:1;O2FA 22:5;O2PA O-31:5FA 22:5;O3PA O-31:4PC 38:8PG 32:2;OLPS 24:1;OPC 17:0_18:1PC 32:5;OCer 18:1;O2/24:1;OSHexCer 38:4;O2BMP 32:1;OST 19:0SHexCer 38:4;O3ST 21:4;O2LPS 20:2PE O-22:0_20:4ST 24:1;O4;TauFA 22:5Cer 18:1;O2/16:0;ONAE 22:1;OPE 18:0_18:2FA 24:0PC 37:0FA 24:2HexCer 30:3;O2FA 24:1SHexCer 40:5;O2PC 37:2Cer 18:0;O2/16:0PC 37:5PA O-34:3PC O-38:3PA 34:2PC O-38:5NAE 20:2;OPC O-38:4PC O-38:7PC O-38:6HexCer 34:4;O3PA O-34:5PA O-34:4LPC 16:0CerP 40:0;O2PE 32:1PE 32:0FA 22:4;OPI 36:1;OSHexCer 38:2;O3FA 22:5;O4HexCer 36:2;O2PC 38:4;OCer 46:0;O3LPE 19:0LPC 22:3;OPE O-42:3Cer 46:0;O4PE O-40:4PS 44:1ST 19:2;OPC 36:4PC 36:5PC 36:2PC 36:8SHexCer 38:3;O3Hex2Cer 34:5;O2Cer 46:1;O4Cer 46:1;O3PE 34:1_20:4SPB 18:3;OLPE 26:1;OCer 34:3;O2ST 27:0;O5LPC 17:1PG 18:0_18:3;OACer 60:2;O2FA 22:5;OST 20:1;O2;SFA 22:6;O4PC 38:5;OFA 22:6;O3PS 38:5;OSHexCer 40:4;O3SHexCer 40:4;O2FA 24:4NAT 24:3LPC 22:2;OPE 18:1_18:2PE O-41:6LPC O-24:1PC 35:2PMeOH 16:0_16:1FA 26:0PMeOH 16:0_16:0ST 26:0;O5PC 35:3PC 38:2;OST 18:1;O2;SCer 40:3;O2PE O-36:6PG 34:2LPC O-18:2FA 15:2PE O-36:3PA O-36:6LPC O-18:1PA O-36:4PE O-36:0PE O-36:1PG 18:1_18:2FA 15:0PC 16:0_20:3PE 34:1PE 34:0PE 34:3PS 38:6PS 38:4PS 38:3PS 38:1SHexCer 36:0;O2PC O-40:5LPC 22:1;OLPE 17:1PC O-40:6PC O-40:4LPE 22:1;OPA 40:3HexCer 40:0;O3HexCer 40:0;O2PC O-40:8LPC 22:4PE 36:1_20:4LPC 22:1PC 34:3PE 18:0_20:4;OPC 34:1PC 34:6PC 34:4PG 34:2;OPC 34:5FA 22:2;O2PC 38:3;OCer 40:0;OFA 20:6;O2Cer 18:0;O3/16:0;OPC 18:0_18:3PC 18:0_18:2PE 16:0_20:5Hex2Cer 32:2;O2PE 16:0_20:4ACer 58:2;O2FA 20:6;O3FA 14:1FA 14:0PE 33:2Cer 36:0;O4Cer 42:3;O4Cer 42:3;O3PE 33:5Hex2Cer 44:3;O2Cer 40:2;O3Cer 40:2;O2Cer 40:2;O4LPE 18:0LPE O-20:0PIP2 34:5FA 26:1LPE 18:1PE O-22:1_18:1LPE O-20:4LPE 22:2;OPC 33:0PE 18:0_20:5;OST 24:1;O3PC 33:3FA 20:5;O3FA 20:5;O2PC 33:1ST 26:3;O2;GlyFA 20:5;O4Cer 42:2;O4Cer 42:2;O3ST 24:1;O4PG 36:3PG 36:2PG 36:4PE O-18:1_20:5LPC O-16:2FA 17:1PE O-34:0PE O-34:1PE O-34:2Hex2Cer 32:4;O2Cer 34:1;O3PE 31:4;OCerP 38:0;O2PC 36:5;OPE 36:2Cer 34:1;O4PE 36:1PG 18:0_20:3PE 36:0PE O-46:7PS 36:4LPC 14:0;OPS 36:2PA 42:0Cer 34:0;ONAE 24:2HexCer 43:2;O3Cer 18:0;O2/16:0;OPE 18:0_20:6;OPC 32:4NAE 18:2;O2PC 32:2PC 32:3FA 16:0;O2PE 46:3;OLPC 20:1;OHexCer 38:3;O2FA 16:2Cer 22:0;O2/18:1;OHexDG 37:2Cer 44:0;O4Cer 44:0;O2PC 36:4;OPS 18:0_20:4PS 18:0_20:3CerP 46:2;O2FA 18:5;OPE 35:2PE O-22:0_22:6FAHFA 34:3;OLPE 16:0LPE 16:1falseHigh-confidence untargeted lipidomics of mammalian serum using LC-TIMS-PASEFThis study aimed to establish and validate a robust untargeted lipidomics workflow for the analysis of human serum using reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry time-of-flight mass spectrometry (RP-LC-TIMS-TOF MS). Commercial pooled human serum was used as a standardized biological matrix to evaluate lipid extraction, chromatographic separation, ion mobility separation, and data-processing performance.2026-05-262026-03-08MTBLS14002