MetaboLights

application/xml

ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/m_MTBLS14048_LC-MS_positive_hilic_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/m_MTBLS14048_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/m_MTBLS14048_LC-MS_negative_reverse-phase_metabolite_profiling-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/m_MTBLS14048_LC-MS_positive_reverse-phase_metabolite_profiling-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/m_MTBLS14048_LC-MS_negative_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/a_MTBLS14048_LC-MS_positive_hilic_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/a_MTBLS14048_LC-MS_negative_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/a_MTBLS14048_LC-MS_positive_reverse-phase_metabolite_profiling-1.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/a_MTBLS14048_LC-MS_positive_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/a_MTBLS14048_LC-MS_negative_reverse-phase_metabolite_profiling-1.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048/s_MTBLS14048.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14048Targeted analysis for aromatic amino acid metabolites (Assay 1)Authentic standards of the aromatic amino acids (AAAs) and derivatives (phenyllactic acid, phenylacetic acid, phenylpropionic acid, tryptamine, indolelactic acid, indolealdehyde, indoleacetic acid, indoleethanol, indolepropionic acid, tyramine, 4-hydroxyphenyllactic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid) were obtained from Sigma-Aldrich. Isotope-labelled aromatic amino acids used as internal standards (L-phenylalanine (ring-d5, 98%), L-tyrosine (ring-d4, 98%), L-tryptophan (indole-d5, 98%) and indoleacetic acid (2,2-d2) of the highest purity grade available were obtained from Cambridge Isotope Laboratories. Metabolite identification was performed by comparison of retention times and accurate mass (5 mDa accuracy) with those of authentic standards analyzed under identical LC-MS conditions. Non-targeted LC-MS/MS analysis for amino acids and related molecules (Assay 2)L-tryptophan, L-phenylalanine, L-leucine, L-isoleucine, L-aspartic acid, L-glutamine, and L-arginine all analytical standard grade (98% purity) were acquired from of Sigma Aldrich (Søborg, Denmark). Individual 5mM stock solutions of these compounds were prepared in Milli-Q water. The stock solutions were then used to prepare a 20 mM mixture 1 (Mix1) and a 10 mM mixture 2 (Mix2), both in Milli-Q water. Mix1 contained L-tryptophan, L-phenylalanine, L-leucine, and L-isoleucine whereas Mix2 contained L-aspartic acid, L-glutamine, and L-arginine. Both mixes were prepared and used for quality control purposes for the non-targeted metabolomics analyses. MZmine 2.53 (Pluskal et al. 2010; Schmid et al. 2023) was utilized to generate a table of features, which was successively manually curated to verify the correct feature ID from automatic open access and in-house library database match (in both MS1 and MS2). Non-targeted LC-MS/MS lipidomics analysis (Assay 3)3β-hydroxy-5-cholestene 3-linoleate (ChoE(18:2)), 1,2,3-Triheptadecanoylglycerol (TG(17:0/17:0/17:0)) and 1,2-dimyristoyl-sn-glycero-3-phospho(choline-d13) PC(14:0/d13) were purchased from Sigma Aldrich (Søborg, Denmark) and 1-stearoyl-2-linoleoyl-sn-glycerol (DG(18:0/20:4)), 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine(LPC(16:0), 1-octadecanoyl-sn-glycero-3-phosphocholine (LPC(18:0)), 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(16:0/18:1)), 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:0p/18:1(9Z))), 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC(18:0p/22:6), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol (PI(18:0/20:4))), N-heptadecanoyl-D-erythrosphingosine (Cer(d18:1/17:0)), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)), 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM(d18:1/17:0)) were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA) whereas Tripalmitin-1,1,1-13C3 (TG(16:0/16:0/16:0)-13C3), Trioctanoin-1,1,1-13C3 (TG(8:0/8:0/8:0)-13C3) from Larodan AB (Solna, Sweden). Individual 1mg mL-1 stock solutions were prepared in MeOH:CHCl3 (1:2, v/v). The stock solutions were then used to prepare the following two standard mixtures in CHCl3:MeOH (2:1, v/v): 10mg mL-1 LIST and 10 mg mL-1 LST. The LIST mixture, which was used as internal standard, consisted of Cer(d18:1/17:0), LPC(17:0), PC(14:0/d13), PC(16:0/d31/18:1), PC(17:0/17:0), PE(17:0/17:0), SM(d18:1/17:0), TG(16:0/16:0/16:0)-13C3 and TG(8:0/8:0/8:0)-13C3). The LST mixture, which was used for quality control purposes, consisted of ChoE(18:2), DG(18:0/20:4), LPC(16:0), LPC(18:0), PC(16:0/18:1), PC(18:0p/18:1(9Z)), PC(18:0p/22:6), PI(18:0/20:4), TG(17:0/17:0/17:0) and it was used for quality control purposes. Compound Discoverer (version 3.3 sp3, Thermo Fisher Scientific Co.) was utilized to generate a table of features, which was successively manually curated to verify the correct feature ID from automatic open access and in-house library of lipids database match (in both MS1 and MS2). Furthermore, lipid compounds are reported at a species level. Targeted analysis for bile acids (Assay 4)Bile acid standards were acquired from Sigma-Aldrich (Sweden, TCA, TUDCA, TCDCA, TDCA, TLCA, GCA, GCDCA, GDCA, GLCA, CA, UDCA, CDCA, DCA, LCA), CDN isotopes (Quebec, Canada, d4-GCDCA, d4-GLCA, d4-GCA, d4-GUDCA, d4-CDCA, d4-UDCA, d4-LCA) and Toronto Research Chemicals (Downsview, Ontario, Canada, d4-CA, d4-TCA). The quantification was made using external standard curves and data was processed using MuliQuant software (Sciex, Concord, Canada).MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - hilicLiquid Chromatography MS - positive - reverse-phaseTargeted analysis for aromatic amino acid metabolites (Assay 1)Aromatic amino acid metabolites in plasma were analyzed using pooled plasma as quality control by an ultra-performance liquid chromatography system consisting of a Dionex Ultimate 3000 RS liquid chromatograph (Thermo Scientific Inc., Waltham, Massachusetts, USA). The analytes were separated on a Poroshell 120 SB-C18 column with a dimension of 2.1 × 100 mm and 2.7 µm particle size (Agilent Technologies, Santa Clara, USA using water with 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B). Quality control samples and standard mixtures were analyzed both before and after the batch of samples, as well as after every tenth sample. Non-targeted LC-MS/MS analysis for amino acids and related molecules (Assay 2 and 3)Amino acids and related molecules were analysed using a Vanquish Duo UHPLC binary system coupled to an Orbitrap IDXTM mass spectrometer (Thermo Fisher Scientific Co., Waltham, Massachusetts, USA). Two different chromatographic methods were used to improve chemical coverage: 1. An ACQUITY UPLC BEH Amide (100 × 2.1 mm, 1.7 mm particle size (WatersTM, Milford, MA, USA) column equipped with an ACQUITY UPLC BEH amide guard was used for the chromatographical separation (Assay 2). The column was kept at 40 °C throughout the analysis. The mobile phase consisted of MilliQ water + 0.1% HCOOH (A), and ACN + 0.1% HCOOH (B). The following gradient elution was used at a flow rate of 0.35 mL min–1: 0-0.8 min 85%, 0.8-3.2 min 85% to 50% B, 3.2-4.2 min 50% B, 4.2-5.2 min 50% to 30% B. The column was re-equilibrated for 3 min at 85% B (Nieto-Domínguez et al. 2024). The autosampler temperature was kept at 7 °C and the injection volume was 1 mL, 2. A ACQUITY BEH C18 (100 × 2.1 mm, 1.7 μm particle size, Waters) column equipped with an ACQUITY BEH C18 guard column was used for the chromatographical separation (Assay 3). The column was kept at 40 °C throughout the analysis. The mobile phase consisted of MilliQ water + 0.1% HCOOH (A) and ACN + 0.1% HCOOH (B). The following gradient elution was used at a flow rate of 0.35 mL min–1: 0-0.8 min 2% B, 0.8-3.3 min 2% to 5% B, 3.3-10 min 5% to 100% B, 10-11 min 100% B. The column was then re-equilibrated at 2% B for 2.7 min[39]. The autosampler temperature was kept at 7 °C and the injection volume was 1 mL. Non-targeted LC-MS/MS lipidomics analysis (Assay 3)Non-targeted lipidomics analyses were performed using a Vanquish UHPLC binary system coupled to an Orbitrap FusionTM mass spectrometer (Thermo Fisher Scientific Co., Waltham, Massachusetts, USA). The chromatographical separation was performed as described previously (O'Gorman et al. 2017) using an ACQUITY BEH C18 (100 × 2.1 mm, 1.7 μm particle size) (WatersTM) column equipped with an ACQUITY BEH C18 guard column (maintained at 50°C throughout the analyses). The mobile phase consisted of MilliQ water + 0.1% HCOOH + 1% 1M NH4Ac (v/v/v) (A) and ACN:IPA (1:1, v/v) + 0.1% HCOOH + 1% 1M NH4Ac (v/v/v) (B). The flow rate was 0.4 mL min-1 and the injection volume was 2 mL and the samples were kept at 10 °C in the autosampler prior to analysis. The gradient elution was as follows: 0 to 2 min 35% B to 80% B, 2 to 7 min 80% B to 100% B and 7 to 14 min 100% B. The column was then re-equilibrated at 35% B in 7 min. Targeted analysis for bile acids (Assay 4)Bile acids were analyzed using a Waters ACQUITY Ultra-Performance Liquid Chromatography (UPLC) Premiere System (Waters Corporation, Milford, MA, USA) coupled to a Waters TQ-Absolute mass spectrometer (Waters Corporation, Milford, MA, USA) and according to previous work (Tremaroli et al. 2015). The samples were injected (5 µL) and bile acids were separated on a Kinetex C18 column (1.7µm, 2.1 x 100 mm; Phenomenex, USA) using water with 7.5 mM ammonium acetate and 0.019% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B).Multi-omics analysis reveals microbiota-metabolite crosstalk in MASLD pathogenesis in a non-obese murine model.DTU National Food InstituteAndreas Koulouktsisblood plasmamixtureliversolventmass spectrometry assayTargeted analysis for aromatic amino acid metabolites (Assay 1)Extraction and profiling of aromatic amino acid (AAA) metabolites in circulating plasma from each group (n = 6 per group) was performed as described earlier (Zhu et al. 2011; Laursen et al. 2021). After thawing at room temperature, the internal standard mix (L-phenylalanine (ring-d5, 98%), L-tyrosine (ring-d4, 98%), L-tryptophan (indole-d5, 98%); 4 μg mL-1) was added to plasma samples (1:4, v/v). An equal volume of 0.1% formic acid was added to plasma, followed by vortexing and addition of 400 μL of cold methanol. After vortexing, samples were incubated at -20 °C for at least 1 h, for protein precipitation. To generate clear extracts, the samples were centrifuged twice at 16,000g at 4 °C for 10 min and dried at 40 °C under nitrogen gas. Reconstitution was performed in 40 μL Milli-Q water. The samples were then centrifuged at 5,000g at 4 °C for 5 min and the clear extract was transferred to vials with inserts prior to analysis. Non-targeted LC-MS/MS analysis for amino acids and related molecules (Assay 2)Samples for the analysis were extracted by pipetting equal volumes (35 μL) of circulating plasma from each group (n = 6 per group) and by transferring these into Eppendorf tubes. The samples were centrifuged at maximum speed for 5 min at 4 °C. A 25 μL aliquot of the resulting supernatant was transferred to a new tube and mixed with a 6:4 MeOH:H2O solution (1:4 sample-to-solvent ratio). The mixture was shaken at 1000 rpm for 3 minutes and subsequently centrifuged again (maximum speed, 5 minutes, 4 °C). An equal volume (~100 μL) of the supernatant was collected and transferred into a fresh Eppendorf tube. This extraction procedure was repeated once after which the supernatants were combined and dried at 30 °C using an Eppendorf® Concentrator Plus. All samples were reconstituted in MeOH:H2O (80:20, v/v) prior to analysis.  Non-targeted LC-MS/MS lipidomics analysis (Assay 3)For the sample preparation, 10 mL of 0.9% NaCl and 28 mL of LIST mixture were added to 10 mL of the serum samples (n = 6 per group) after which the samples were diluted 1:1(v/v) with CHCl3:MeOH (2:1, v/v). Each sample was then vortex mixed for 2 minutes and incubated on crushed ice for 30 min. Finally, the samples were centrifuged at 4 °C for 3 min at 10,000 rpm and the lower phase for each sample was collected and further diluted 1:1 (v/v) using CHCl3:MeOH (2:1, v/v). Targeted analysis for bile acids (Assay 4)Plasma samples (25-50 µL) from Chow (n = 5) and CDAHFD-fed (n = 6) mice were extracted with 10 volumes of methanol:water (1:1, v/v) containing deuterated internal standards (d4-CA, d4-TCA, d4-GCA, d4-GCDCA, d4-GUDCA, d4-GLCA, d4-UDCA, d4-CDCA, d4-LCA; 50 nM of each) (Tremaroli et al. 2015). Chow group exhibits lower size due to insufficient portal vein blood from one animal. After 10 minutes of vortex and 10 minutes of centrifugation at 20000g, the supernatant was evaporated under a stream of nitrogen and reconstituted in 100-200 µL methanol:water (1:1, v/v). Tissue samples (20-50 mg) were homogenized in 500 µL methanol, with 2.5 µM of the internal standards, using a Precellys homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). After centrifugation at 20000g for 10 minutes the supernatant was diluted 10-50 times in methanol:water (1:1, v/v).Mus musculusSolvent Blankreference compoundhttps://www.ebi.ac.uk/metabolights/MTBLS14048Martin Laursen. Technical University of Denmark. mfrla@food.dtu.dk.Anna Hassing. University of Copenhagen. anna.hassing@sund.ku.dk.Mikael Pedersen. Technical University of Denmark. mikp@food.dtu.dk.Marcus Henricsson. University of Gothenburg. marcus.henricsson@astrazeneca.com.Julius Brinck. Technical University of Denmark. juem@food.dtu.dk.Angel Phanthanourak. Technical University of Denmark. anglup@biosustain.dtu.dk.Andreas Koulouktsis. Technical University of Denmark. andkou@dtu.dk.Fredrik Bäckhed. University of Gothenburg. fredrik.backhed@wlab.gu.se.Juliana Assis. Technical University of Denmark. jasge@dtu.dk.Ditte Lützhøft. Technical University of Denmark. dlutzhoft@zealandpharma.com.Alberto Santos. Technical University of Denmark. albsad@biosustain.dtu.dk.Antonios Otapasidis. Technical University of Denmark. s232996@student.dtu.dk.Ruben Vazquez-Uribe. VIB-KU Leuven Center for Microbiology. ruben.vazquezuribe@kuleuven.be.Morten Sommer. Technical University of Denmark. msom@dtu.dk.Linda Ahonen. Technical University of Denmark. linaho@biosustain.dtu.dk.Jens Jacobsen. University of Copenhagen. jcbrings@sund.ku.dk.Jonas Treebak. University of Copenhagen. jttreebak@sund.ku.dk.Albert Pallejà. Technical University of Denmark. apca@biosustain.dtu.dk.Daniela Rago. Technical University of Denmark. danrag@biosustain.dtu.dk.Targeted analysis for aromatic amino acid metabolites (Assay 1)Data processing was performed using QuantAnalysis v.2.2 (Bruker Daltonics, Bremen, Germany), employing calibration curves based on linear regression for each metabolite. These curves were generated by plotting the ratio of analyte peak areas to those of the internal standard against the known concentrations of the calibration standards. Non-targeted LC-MS/MS analysis for amino acids and related molecules (Assay 2)MZmine 2.53 (Pluskal et al. 2010; Schmid et al. 2023) was utilized to generate a table of features, which was successively manually curated to verify the correct feature ID from automatic open access and in-house library database match (in both MS1 and MS2). Raw data for the present analysis were not transformed. Non-targeted LC-MS/MS lipidomics analysis (Assay 3)Compound Discoverer (version 3.3 sp3, Thermo Fisher Scientific Co.) was utilized to generate a table of features, which was successively manually curated to verify the correct feature ID from automatic open access and in-house library of lipids database match (in both MS1 and MS2). Furthermore, lipid compounds are reported at a species level. Raw data for the present analysis were not transformed. Targeted analysis for bile acids (Assay 4)The quantification was made using external standard curves and data was processed using MuliQuant software (Sciex, Concord, Canada).GroupDescriptionMetabolite profilingandkou@dtu.dkThe animal study was conducted in agreement with the Danish Animal Experiments Act on protection of animals used for scientific purposes (LBK 1107 from 02/07/2022) and the EU directive 2010/63/EU of the European Parliament. The study was approved by the Danish Animal Experiments Inspectorate under the Ministry of Food, Fishing and Agriculture (license number 2021-15-0201-00925). Male C57BL/6NTac mice (5 weeks of age) were obtained from Taconic Biosciences and co-housed three mice per cage in a controlled environment (22 °C ± 1 °C, humidity 55% ± 5%) with a 12h light/dark cycle. The average and variance of baseline body weight were used to stratify the experimental groups (n = 6 per group) after a 2-week acclimatization period on Chow diet (A30, Safe diets). During the 12-week study period mice were fed with either a choline-deficient L-amino acid diet with 60 kcal% Fat and 0.1% methionine (A06071302, Research Diets, Inc.) for disease induction or a regular Chow diet. Body weight was monitored on a weekly basis, while faecal samples were collected biweekly in PBS (pH 7.4; GibcoTM). Upon completion (12 weeks) and after 5 h of fasting, animals were anaesthetized with isoflurane (Fresenius Kabi), heart and portal blood was collected, and the animal were euthanized by cervical dislocation. Blood was mixed with a protease inhibitor cocktail (cOmpleteTM; Roche) and a DPP4 inhibitor (sitagliptin; Sigma-Aldrich) in a Microtainer® PSTTM LH blood collection tube (Becton Dickinson). Plasma was obtained according to the vendor, aliquoted and stored at -80 °C until further analysis. Liver weight and colon length were recorded, and tissues were frozen immediately in liquid nitrogen and stored at -80 °C. Intestinal (cecum, small and large intestine) contents were collected in PBS solution in pre-weighted Eppendorf tubes and stored at -80 °C for further metagenomic analysis.  ReferencesLaursen, M.F., Sakanaka, M., von Burg, N. et al. Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut. Nat Microbiol 6, 1367–1382 (2021). https://doi.org/10.1038/s41564-021-00970-4Nieto-Domínguez, M., Sako, A., Enemark-Rasmussen, K. et al. Enzymatic synthesis of mono- and trifluorinated alanine enantiomers expands the scope of fluorine biocatalysis. Commun Chem 7, 104 (2024). https://doi.org/10.1038/s42004-024-01188-1O Gorman, A., Suvitaival, T., Ahonen, L. et al. Identification of a plasma signature of psychotic disorder in children and adolescents from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. Transl Psychiatry 7, e1240 (2017). https://doi.org/10.1038/tp.2017.211Pluskal, T., Castillo, S., Villar-Briones, A. et al. Mzmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics 11, 395-406 (2010). https://doi.org/10.1186/1471-2105-11-395Radi, M.S., Munro, L.J., Rago, D. and Kell, D.B. An untargeted metabolomics strategy to identify substrates of known and orphan E. coli transporters. Membranes 14, 70 (2024). https://doi.org/ 10.3390/membranes14030070Schmid, R., Heuckeroth, S., Korf, A. et al. Integrative analysis of multimodal mass spectrometry data in MZmine 3. Nat Biotechnol 41, 447–449 (2023). https://doi.org/10.1038/s41587-023-01690-2Tremaroli, V., Karlsson, F., Werling, M., et al. Roux-en-Y gastric bypass and vertical banded gastroplasty induce long-term changes on the human gut microbiome contributing to fat mass regulation. Cell Metab 22, 228-238 (2015). https://doi.org/10.1016/j.cmet.2015.07.009Zhu, W., Stevens, A.P., Dettmer, K. et al. Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 401, 3249–3261 (2011). https://doi.org/ 10.1007/s00216-011-5436-yMetabolomicsPlasmaQTRAP 5500MRM/SRMTryptophanLipidomicsOrbitrapAmino Acidstargeted metabolitesaromatic amino acidIndole Alkaloidsbile acidsuntargeted metabolitesUltra Performance Liquid Chromatography - Mass Spectrometerultra high-performance liquid chromatographMetabolic Dysfunction-Associated Steatotic Liver DiseasePlasmaQTRAP 5500MRM/SRMTryptophanLipidomicsOrbitrapAmino Acidstargeted metabolitesaromatic amino acidIndole Alkaloidsbile acidsuntargeted metabolitesUltra Performance Liquid Chromatography - Mass Spectrometerultra high-performance liquid chromatographMetabolic Dysfunction-Associated Steatotic Liver DiseaseTargeted analysis for aromatic amino acid metabolites (Assay 1)Aromatic amino acid metabolites in plasma were analyzed using pooled plasma as quality control by an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry system consisting of a Dionex Ultimate 3000 RS liquid chromatograph (Thermo Scientific Inc., Waltham, Massachusetts, USA) coupled to a Bruker maXis time-of-flight mass spectrometer equipped with an electrospray interphase (Bruker Daltonics, Massachusetts, USA) operating in negative mode (Zhu et al. 2011; Laursen et al. 2021). The analytes were separated on a Poroshell 120 SB-C18 column with a dimension of 2.1 × 100 mm and 2.7 µm particle size (Agilent Technologies, Santa Clara, USA using water with 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B). Quality control samples and standard mixtures were analyzed both before and after the batch of samples, as well as after every tenth sample. Non-targeted LC-MS/MS analysis for amino acids and related molecules (Assay 2 and 3)The MS/MS measurements were performed using an Orbitrap IDXTM mass spectrometer (Thermo Fisher Scientific Co., Waltham, Massachusetts, USA) in positive-heated electrospray ionization (HESI) mode with a voltage of 3500 acquiring in full MS/MS spectra (data dependent acquisition-driven MS/MS) with a m/z range of 70–1000 and in profiling mode. The MS1 resolution was set at 120,000 and the MS2 resolution was set at 30,000. Precursor ions were fragmented by stepped high-energy collision dissociation (HCD) using collision energies of 20, 40 and 55 eV. The automatic gain control (AGC) target value was set at 4×105 for the full MS and 5×104 for the MS/MS spectral acquisition. A blank (Milli-Q water) sample as well as Mix1 and Mix2 were analysed throughout the sample set to check for mass error, retention time (RT) shift and overall signal reproducibility (defined as area under the curve, AUC). The results show high-quality data where the predefined acceptance criteria (< 5ppm for mass accuracy, 0.1 min for RT shift and <15% for AUC reproducibility) for these parameters were fulfilled. Non-targeted LC-MS/MS lipidomics analysis (Assay 3)The MS/MS measurements were performed in positive-heated electrospray ionization (HESI) mode with a voltage of 3500 acquiring in full MS/MS spectra (data dependent acquisition-driven MS/MS) with a m/z range of 100–1100, similarly as mentioned above for the metabolomics analysis. The LST mixture was analysed throughout the sample set to check for mass error and retention time shift. Both parameters were found to fulfil the predefined acceptance criteria: < 5ppm for mass accuracy and 0.1 min for RT shift. The LIST mixture was utilized to assess sample preparation and overall signal reproducibility (expressed as AUC). The predefined acceptance criteria of %CV < 35 for biological samples was achieved throughout the sample set. Targeted analysis for bile acids (Assay 4)Bile acids were analyzed using a Waters ACQUITY Ultra-Performance Liquid Chromatography (UPLC) Premiere System (Waters Corporation, Milford, MA, USA) coupled to a Waters TQ-Absolute mass spectrometer (Waters Corporation, Milford, MA, USA) and according to previous work (Tremaroli et al. 2015). The MS/MS measurements were performed in a negative electrospray ionization on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode over an m/z range of 300-600.falseMulti-omics analysis reveals microbiota-metabolite crosstalk in MASLD pathogenesis in a non-obese murine modelThe rising global prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD), including its severe form metabolic dysfunction-associated steatohepatitis (MASH), underscores the urgency to elucidate its pathophysiology. To investigate key metabolic interactions central to MASLD progression between the gut microbiota and the host, this study employed a mouse model based on a choline-deficient, amino acid-defined, high-fat diet (CDAHFD). CDAHFD-fed mice rapidly developed severe MASLD, evidenced by hepatomegaly, inflammation and fibrosis, accompanied by significant shifts in certain gut microbial taxa. Metagenomic analysis revealed functional shifts towards pathways involving aromatic amino acid metabolism, particularly tryptophan metabolism. Targeted analysis of circulating metabolites further demonstrated that these shifts correlated with decreased microbial indole derivatives and increased host-derived kynurenine, strongly associated with disease severity. Bile acid profiling identified substantial increases in conjugated primary bile acids, particularly tauro-β-muricholic acid, linked to impaired gut-liver signaling pathways. Multi-omics network analysis suggested a critical role of Clostridia in metabolizing aromatic amino acids, associated with reduced microbial diversity and exacerbated metabolic disruptions. These findings highlight the integral role of the microbiota in MASLD pathogenesis, suggesting that microbiome-derived secondary metabolites and altered bile acid metabolism may serve as potential diagnostic and therapeutic targets for managing MASLD. 2026-03-132026-03-13MTBLS14048