LC–MS spectral files were processed in Compass MetaboScape 2021b (Bruker Daltonik, Germany). Processing parameters are available (https://link.springer.com/article/10.1007/s11306-025-02309-0, Supplement S6). For MSEA and ORA, metabolites were identified via spectral library search. Three proprietary and one open database were searched hierarchically in the following order: 1. Bruker MetaboBASE Personal Library 3.0 (215,367 spectra of 100,679 compounds), 2. Bruker NIST 2020 MSMS Spectral Library (1,021,914 spectra of 27,840 compounds), 3. MSDial V17 ESI( ±)-MS/MS from authentic standards (obtained from https://systemsomicslab.github.io/compms/msdial/main.html MSP in August 2022, 324,191 spectra of 21,126 compounds and 3848 spectra of 2553 compounds for the positive and negative mode libraries, respectively), 4. Bruker HMDB Metabolite Library 2.0 (6022 spectra of 824 compounds). Only annotations validated by MS/MS spectra were allowed and MS/MS spectra were filtered by precursor m/z. The following maximum tolerances were set: m/z 5.0 ppm, mSigma 120, MS/MS score 200 and collision cross section (CCS) 5.0%. In MetaboScape, mSigma is a score to describe the deviation between the expected and the measured isotope pattern (the lower the better) and the MS/MS score is an arbitrary number between 1000 (perfect match between the compound MS/MS spectrum and the library MS/MS spectrum) and 0 (no match between the compound MS/MS spectrum and the library MS/MS spectrum).
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - reverse phase","Liquid Chromatography MS - positive - hilic","Liquid Chromatography MS - positive - reverse phase","Liquid Chromatography MS - negative - hilic"],"chromatography_protocol":["All chemicals were MS grade. Double distilled water was generated using an in-house distill. For liquid chromatograophy, an Elute UHPLC pump (Bruker Daltonik GmbH, Bremen, Germany) was used. Samples were analyzed with a reversed phase (RP) and a hydrophilic interaction chromatography (HILIC) column. The auto sampler (Elute Autosampler, Bruker Daltonik GmbH, Bremen, Germany) was operated in ul Pickup mode and cooled to 8 °C. Injection volumn was 2 µl and each sample was measured 3 times. QC samples were injected at the beginning, at the end and multiple times during each run. The column oven (Elute Column Oven, Bruker Daltonik GmbH, Bremen, Germany) was set to 35 °C for both methods.
Reversed Phase
For RP measurements, a ZORBAX Eclipse Plus C18 column (Agilent, 2.1 x 50 mm, 1.8 µm) was used with a ZORBAX Eclipse Plus C18 (Agilent, 2.1 x 5 mm, 1.8 µm) as pre column. Solvent A was double distilled water, solvent B acetonitrile, both were acidified with 0.1 % formic acid (0 min 5 % B, 1 min 5 % B, 7.5 min 100 % B, 10 min 100 % B, 10.1 min 5 % B, 15 min 5 % B). Flow rate was 0.3 ml/min.
HILIC
HILIC measurements were carried out using an ACQUITY UPLC BEH Amide column (Waters, 2.1 x 50, 1.7 µm) with a ACQUITY UPLC BEH Amide (Waters, 2.1 x 5 mm, 1.7 µm) as pre column. Solvent A was 90 % double distilled water and 10 % acetonitrile, solvent B was 90 % acetonitrile and 10 % double distilled water. Both contained 10 mM ammonium acetate and pH was adjusted to 9 with ammonia solution (0 min 99 % B, 0.5 min 99 % B, 6 min 60 % B, 8 min 20 % B, 9 min 20 % B, 10 min 99 % B, 15 min 99 % B). Flow rate was 0.4 ml/min.
"],"publication":["Comparison of popular enrichment methods for untargeted in vitro metabolomics. 10.1007/s11306-025-02309-0. PMID:40715905"],"submitter_affiliation":["Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau","RPTU Kaiserslautern-Landau"],"submitter_name":["Simone Stegmüller","Yannik Schermer"],"organism_part":["Hep-G2 cell","buffer"],"technology_type":["mass spectrometry"],"disease":[""],"extraction_protocol":["Cells were lysed using two rounds of freeze and thaw with 5 min at 37 °C under heavy shaking followed by 1 min in liquid nitrogen and two subsequent rounds of sonication on ice for 10 s with 10 s pause in between. 300 µl lysate was transferred to fresh 1.5 ml reaction tubes and 900 µl methanol cooled to -20 °C was added. Samples were incubated for 10 min at 4 °C under heavy shaking and subsequently incubated for 1 h at -20 °C to aid protein precipitation. Samples were centrifuged at 17,000 x g and 4 °C for 15 min and 1 ml of supernatant was transferred to a fresh reaction tube. Solvent was evaporated under a gentle nitrogen stream. 100 µl methanol/acetonitrile/water (2:2:1) was added to the residues. They were dissolved by incubating for 10 min at 10 °C under heavy shaking followed by 10 min sonication on ice in a sonication bath. Samples were centrifuged again at 17,000 x g and 4 °C for 10 min and transferred to a vial for Ultra-High Performance Liquid Chromatography (UHPLC) Mass Spectrometry (MS) analysis. Lysates of untreated Hep-G2 cells served as quality controls (QC).
"],"organism":["blank","Homo sapiens"],"further_data_processing_protocol":["Feature tables were exported to CSV files and further processed in R version 4.4 (R Core Team, 2022) using several libraries (Robinson et al., 2024; Wickham et al., 2019). The code is available on GitHub (https://github.com/yasche/Pathway_comparison_SI). Briefly, features were filtered out if they were also present in blank samples (i.e., their maximum intensity in samples was less than or equal to 3 times the maximum intensity of that feature in blank samples), if they showed poor reproducibility (i.e., their coefficient of variation in quality control samples was larger than 0.2) or if they were not found reliably across samples (i.e., if they were not found in at least 75 % of the samples of either treatment or control group). Missing values for remaining features were replaced with 1/5 the minimum measured intensity for that feature, intensities were normalized sample-wise by dividing them by the sample median (Ramirez et al., 2018). Technical replicates were combined by calculating the mean for each feature. KEGG (Kanehisa et al., 2025) identifiers were mapped to compound names using a combination of compound ID conversion in MetaboAnalyst 6.0 (Pang et al., 2024) and manual search. Feature tables for the same compound obtained with different separation methods and polarities were combined and intensities were log2-transformed. Additional, method-specific preparation steps were performed due to different input requirements.
For ORA, p-values were calculated using two-sided Welch’s t-test. Metabolites were considered significantly changed if p ≤ 0.05 and their change was at least 1.5-fold. For each compound, a reference metabolome containing all annotated metabolites was provided in the pathway analysis to account for method bias (Wieder et al., 2021).
As the MSEA module on MetaboAnalyst 6.0 does not allow for duplicates, duplicate metabolites were removed. For this, t-scores were calculated using log2-transformed intensities. Only the duplicate with the highest absolute t-score was kept and other duplicates were removed.
For Mummichog, p-values and t-scores were calculated based on log2-transformed intensities. The input consisted of m/z, p-values and t-scores.
Enrichment analysis was done using MetaboAnalyst 6.0 web service (Pang et al., 2024). MSEA and ORA were performed using the Enrichment Analysis module, Mummichog using the Functional Analysis module. No additional filtering or normalization was performed. KEGG (Homo sapiens) was used as Metabolite Set/Pathway Library. Only pathways containing > 2 entries were considered. For Mummichog, mixed ion mode was chosen with a mass tolerance of 5 ppm. Primary ions were enforced and p-value cutoff was left at the default setting (top 10 % peaks).
Results from different enrichment analysis methods were compared:
Measures of similarity
Similarities for different methods and compounds were assessed using two approaches. Pairwise Jaccard indices J(A,B) were calculated with J(A,B) = intersect(A,B)/union(A,B) where A and B are significantly enriched pathways (p ≤ 0.05) for the two methods or compounds. Additionally, Spearman’s correlation coefficient ρ was calculated for each combination of methods and compounds.
Assessment of the correctness of the results for different enrichment methods
To assess the correctness of the results, the lists of enriched pathways were compared to a list of expected pathways for each compound. To obtain the list of expected pathways, the KEGG database (Kanehisa et al., 2025) was searched for the molecular target of each applied compound. Pathways associated with each target were downloaded and filtered to only contain those present in the model for MSEA, ORA or Mummichog. As a measure of correctness, the share of expected pathways correctly identified amongst the top n (1 ≤ n ≤ 20) enriched pathways was calculated for each enrichment method and compound. For example, 3-bromopyruvic acid’s primary target is hexokinase. This enzyme is associated with 6 pathways present in the model for MSEA, ORA or Mummichog. Of these 6 pathways, Mummichog was able to identify 2 if the top 10 most enriched pathways are considered, corresponding to 2/6 or approximately 33 % of the expected pathways correctly identified.
References
Kanehisa, M. et al. (2025) ‘KEGG: biological systems database as a model of the real world’, Nucleic Acids Research, 53(D1), pp. D672–D677. Available at: https://doi.org/10.1093/nar/gkae909.
Pang, Z. et al. (2024) ‘MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation’, Nucleic Acids Research, 52(W1), pp. W398–W406. Available at: https://doi.org/10.1093/nar/gkae253.
R Core Team (2022) R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. Available at: https://www.R-project.org/.
Ramirez, T. et al. (2018) ‘Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells’, Archives of Toxicology, 92(2), pp. 893–906. Available at: https://doi.org/10.1007/s00204-017-2079-6.
Robinson, D., Hayes, A. and Couch, S. (2024) broom: Convert Statistical Objects into Tidy Tibbles. Available at: https://CRAN.R-project.org/package=broom.
Wickham, H. et al. (2019) ‘Welcome to the Tidyverse’, Journal of Open Source Software, 4(43), p. 1686. Available at: https://doi.org/10.21105/joss.01686.
Wieder, C. et al. (2021) ‘Pathway analysis in metabolomics: Recommendations for the use of over-representation analysis’, PLOS Computational Biology. Edited by K.R. Patil, 17(9), p. e1009105. Available at: https://doi.org/10.1371/journal.pcbi.1009105.
"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS12332"],"author":["Elke Richling. RPTU Kaiserslautern-Landau. Gottlieb-Daimler-Straße, 67663 Kaiserslautern. elke.richling@chem.rptu.de.","Frederic Wagner.","Yannik Schermer. RPTU Kaiserslautern-Landau. Gottlieb-Daimler-Straße, 67663 Kaiserslautern. yannik.schermer@chem.rptu.de.","Simone Stegmüller. RPTU Kaiserslautern-Landau. Gottlieb-Daimler-Straße, 67663 Kaiserslautern. simone.stegmueller@chem.rptu.de."],"data_transformation_protocol":["Raw LC-MS files were processed in Bruker Compass MetaboScape 2021b (Bruker Daltonik GmbH, Bremen, Germany). All raw data files obtained with the same separation and MS method were loaded into Bruker Compass MetaboScape together. Samples were assigned to groups, according to the compound they were treated with, blank, vehicle control or QC. Features were filtered out if they were not present in at least 75 % of the samples of at least 1 group.
MS2 spectrum import was enabled, m/z range was set to 50 - 1500. Chromatograms were cropped after 10 min or 9 min for RP and HILIC, respectively. Seed intensity threshold was set to 1000 and minimum cluster size to 75. Minimum Seed cluster size was set to 100. For chromatographic alignment, the mobility delta was set to 0.05, the m/z delta to 0.015. Recursive feature extraction was enabled. Features were grouped if they did not exceed these thresholds: mobility 0.05, m/z 0.015, retention time 10 s. Ion deconvolution was performed with EIC correlation set to 0.8 and the following ions: Reversed phase positive, primary ion [M+H]+, common ion [M+H-H2O]+, seed ions [M+Na]+ and [M+K]+; Reversed phase negative, primary ion [M-H]-, common ion [M-H-H2O]-, seed ion [M+Cl]-; HILIC positive, primary ion [M+H]+, common ion [M+H-H2O]+, seed ions [M+Na]+, [M+K]+ and [M+NH4]+; HILIC negative, primary ion [M-H]-, common ion [M-H-H2O]-, seed ions [M+Cl]- and [M+CH3CO2]-.Within-batch correction was performed with QC samples. LOESS was used as correction algorithm with a bandwidth of 0.7, post-correction variance threshold of 20 and pre-correction variance threshold of 40. Mass recalibration was performed with the default Na formate positive or negative ion list.
"],"study_factor":["Treatment","Replicate","Batch","Injection","Concentration"],"submitter_email":["yannik.schermer@chem.rptu.de","simone.stegmueller@chem.rptu.de"],"sample_collection_protocol":["Material and Chemicals
Compounds for cell treatment were obtained from various suppliers: FCCP, phenantrene-9,10-dione, 5-fluorouracil, trifluorothymidine, 3-bromopyruvate, mevastatin, simvastatin and 2-deoxyglucose were obtained from BLD Pharmatech GmbH (Reinbek, Germany), metrizamide and menadione from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany), and antimycin A from Thermo Fisher Scientific (Darmstadt, Germany). All compounds have a purity of ≥ 95 % and identity was confirmed via 1H NMR (Shield, Bruker Corporation, Billerica, MA, USA). DMSO-d6 and deuterium oxide for NMR analysis were obtained from Deutero GmbH (Kastellaun, Germany).
Cell Culture Conditions
Hep-G2 cells were cultivated in RPMI 1640 medium (Gibco) with 10 % fetal bovine serum (FBS), qualified, Brazil (Gibco) and 1 % penicillin streptomycin (PS) at 37 °C and 5 % CO2 and split with a 1:5 or 1:4 ratio when approximately 80 % confluency was reached using trypsin.
Dose-finding
For dose finding via resazurin reduction assay, compounds were dissolved in DMSO, except for 3-bromo pyruvate which was dissolved in double distilled water to prevent decay. Various dilutions were prepared. Dilutions were mixed with RPMI 1640 medium, supplemented with 5 % FBS and 1 % PS. Final DMSO concentration was 0.5 %. 200 µl suspension containing 60,000 cells was put in each well of a 96 well plate and incubated for approximately 24 h under cell culture conditions. Medium was removed, and cells were incubated with 200 µl compound mixture for 2 h. Vehicles served as negative controls while 0.1 % SDS served as a positive control. After 2 h cells were washed with 200 µl PBS (37 °C). RPMI 1640 medium containing 1 % PS and 10 % resazurin solution was added and cells were incubated for an additional hour. Finally, fluorescence was measured (37 °C, 544 nm excitation, 590 nm emission using a BioTek Synergy H1 microplate reader from Agilent (Santa Clara, CA, USA)).
Dose-response modelling was done in R version 4.4 (R Core Team, 2022) using the drc package (Ritz et al., 2015). First, blanks were subtracted, and values were normalized to their vehicle control. Next, a four-parameter log-logistic model was fitted and the concentration which causes a 10 % decrease in viability (i.e., the IC10) was derived. The code for the analysis is available on GitHub (https://github.com/yasche/Pathway_comparison_SI). Estimated IC10 values are given below:
Antimycin A 94.4 µM
3-Bromopyruvic acid 78.9 µM
FCCP 53.4 µM
Menadione 36.8 µM
Mevastatin 77.5 µM
Phenanthrene-9,10-dione 24.8 µM
Simvastatin 43.0 µM
2-Deoxy-d-glucose* >1000.0 µM
5-Fluorouracil* >1000.0 µM
Metrizamide* >1000.0 µM
Trifluorothymidine* >1000.0 µM
*No reduction in cell viability was observed for up to 1000 µM. For metabolome analysis, cells were therefore treated with 1000 µM of the respective compound.
Cell Treatment and Sample Preparation for Endometabolome Analysis
The protocol for endometabolome analysis was modified after Bi et al. (2013). 6 ml of a suspension containing 4 million cells were seeded in 60 mm dishes and cells were incubated for approximately 24 h under cell culture conditions. Dishes containing 6 ml PBS without cells served as a process blank. Compounds were prepared as described in the section above. Final concentrations in the medium were the calculated IC10 values for the respective compound (see above). After 24 h, medium was removed, and cells were incubated with 6 ml medium containing compound or vehicle control for 2 h at 37 °C and 5 % CO2. Treatment was carried out in quadruplicates for each compound, vehicle control and process blank. To avoid systematic error, a new vehicle control and process blank was done for each batch. Medium was removed and cells were washed with 2 ml PBS pre warmed to 37 °C, twice. Metabolism was quenched by adding 400 µl double distilled water cooled to 4 °C containing protease inhibitor (Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)), quickly followed by liquid nitrogen. Cells were transferred to 1.5 ml reaction tubes using a cell scraper and were immediately flash frozen in liquid nitrogen.
References
Bi, H. et al. (2013) ‘Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells’, Analytical and Bioanalytical Chemistry, 405(15), pp. 5279–5289. Available at: https://doi.org/10.1007/s00216-013-6927-9.
R Core Team (2022) R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. Available at: https://www.R-project.org/.
Ritz, C. et al. (2015) ‘Dose-Response Analysis Using R’, PLOS ONE. Edited by Y. Xia, 10(12), p. e0146021. Available at: https://doi.org/10.1371/journal.pone.0146021.
"],"omics_type":["Metabolomics"],"study_design":["Toxicology","Hep-G2 cell","In Vitro","untargeted metabolites","Pharmacology","Ultra High-performance Liquid Chromatography/Tandem Mass Spectrometry"],"curator_keywords":["Toxicology","Hep-G2 cell","In Vitro","untargeted metabolites","Pharmacology","Ultra High-performance Liquid Chromatography/Tandem Mass Spectrometry"],"mass_spectrometry_protocol":["UHPLC-MS measurements were carried out using an Elute UHPLC pump (Bruker Daltonik, Germany) coupled to a timsTOF Pro (Bruker Daltonik, Germany). Each sample was analyzed in reversed phase (RP) and hydrophilic interaction chromatography (HILIC) positive and negative mode. For RP measurements, a ZORBAX Eclipse Plus C18 column (Agilent, USA, 2.1 × 50 mm, 1.8 µm) was used. HILIC measurements were carried out using an ACQUITY UPLC BEH Amide column (Waters, USA, 2.1 × 50, 1.7 µm). Ion mobility and mass accuracy were calibrated according to the instrument manufacturer prior to each measurement series. Additionally, mass accuracy was calibrated during each measurement using a 10 mM sodium formate (Thermo Fisher Scientific, USA) solution in water/isopropyl alcohol (Honeywell Specialty Chemicals Seelze, Germany) (1:1). The MS was operated in Parallel Accumulation–Serial Fragmentation (PASEF) mode. The mass range was set to m/z 20–1,300 with 1/K0 0.45–1.45 Vs/cm2 and 100 ms ramp time. End plate offset of the ESI source was 500 V with a capillary voltage of 4500 V or 3600 V for positive and negative ion mode, respectively. Nebulizer was set to 2.2 bar and dry gas flow was 9 l/min. Dry temperature was 220 °C. PASEF settings were as follows: Absolute collision energies were 20 eV to 50 eV. Active exclusion was enabled, and exclusion time was 6.0 s with the option to reconsider the precursor if the intensity doubled enabled. The number of PASEF scans was two and the intensity threshold was 100. Each sample was injected three times. QC samples were injected before, during and after sample measurements to allow for within-batch correction.
"],"metabolite_name":["Thr-Phe","Uridine Monophosphate","(2-Acetyloxy-3-hexadecoxypropyl) 2-(trimethylazaniumyl)ethyl phosphate","Diisopropyl phthalate","2-Amino-6-methylbenzonitrile","N-[2-(5,6-Dimethyl-1h-Benzimidazol-2-Yl)ethyl]-4-(4h-1,2,4-Triazol-4-Yl)benzamide","1-(Cyclopropylsulfonyl)piperidine-4-carboxylic acid","N1,N12-Diacetylspermine","2-Ethyl-4-[(z)-2-[(3-formamido-2-hydroxybenzoyl)amino]but-2-enoyl]oxy-3-(2-methylbutanoyloxy)pentanoic acid","1,4-Dioxacycloheptadecane-5,17-dione","3-Phenyl-N-{1-[5-(pyrrolidin-1-YL)pyrimidin-4-YL]-1H-pyrazol-4-YL}propanamide","Phe-ala","2-((3-Methoxyphenyl)sulfanyl)acetic acid","5-Ethyl-2-methylpyridine","4-Tert-Butylaniline","2-(Ethyl(3-methylphenyl)amino)acetonitrile","Heptapropylene glycol","(-)-Rasfonin","Mevinolinic Acid","2,3-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,14,15,17-decahydro-1H-cyclopenta[a]phenanthrene-12,16-dione","19-Aldoandrostenedione","1,5-Naphthalenediamine","Benzenesulfonic acid, 3-amino-4-ethyl-","N,N-Dimethyloctanamide","2-Methylbutyroylcarnitine","N1-Acetylspermidine","Ser-Tyr","Flavin-Adenine Dinucleotide","N-cyclohexyl-4-ethoxypiperidine-1-carboxamide","1-(Pyridin-4-yl)piperidin-4-amine","Phenolsulfonphthalein","9-Fluorenone","2-(4-Morpholinyl)benzothiazole","1-(4-Aminobenzylidene)indene","Artemisinin","Koninginin A","Latanoprost ethyl amide","5-alpha-Dihydrotestosterone glucuronide","Pantothenic Acid","Penicillin G","S-Methoprene","1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine","1-Docosanamine","9-OxoOTrE","Lauryldiethanolamine","Acetyl-leucyl-leucyl-argininal","Val-Ile","Arginine","Linoleamide","3-[1-(4-Fluorobenzenesulfonyl)piperidin-3-yl]propanoic acid","Hydroxyindoleacetic Acid","3-[4-(Pyridin-2-YL)piperazine-1-carbonyl]-4,5,6,7-tetrahydro-1,2-benzoxazole","5,6-Dimethylbenzimidazole","Aureonitol","Glutathione","N1-acetylspermine","Phenylalanylserine","Niacinamide","Diisopropylaniline","trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane","6-Methylquinoline","1-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine","1-Palmitoyl-sn-glycerol","gamma-Glu-leu","Hydrocortisone Acetate","Indole-4-carboxaldehyde","Levocarnitine","N-{1-[(6-ethoxy-3-pyridyl)methyl]tetrahydro-1H-pyrrol-3-yl}-2-(3-pyridyl)acetamide","4,4,7a-trimethyl-3a,5,6,7-tetrahydro-3H-indene-1-carboxylic acid","Cycloxydim-TP BH 517-TSO E/Z-isomer","Tetraethylene glycol monododecyl ether","1-Myristoyl-sn-glycero-3-phosphocholine","Biotin sulfone","Leu-Asp-Cys","Adenosine-5'-diphosphate","Pentaethylene glycol","16alpha,17alpha-Epoxypregnenolone acetate","Oleamide","Cinnamic Acid","1-Cyclohexyl-3-((1-(hydroxymethyl)cyclopropyl)methyl)urea","Benzphetamine","Formestane","Nadide","2-(Methylthio)benzothiazole","Diphenyl sulfone","Octapropylene glycol","2-Amino-1-hydroxyoctadec-4-en-3-one","[4-phenyl-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl]methanamine","16-Methylheptadecanoic Acid","Palmitoyl Carnitine","Lefetamine","N,N-Bis(2-hydroxyethyl)dodecanamide","trans-4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid","Oleoylcarnitine","Pentadecylamine","Hippuric Acid","Uridine-5'-Diphosphate","Oxiglutatione","13-Docosenamide","Ile-Glu-Ala","Phe-Glu","N-[(3S)-Tetrahydro-2-oxo-3-furanyl]octadecanamide","Tyrosine","2-(Thiophen-3-ylmethyl)propanedioic acid","Pravastatin lactone","15d-Prostaglandin A2","9-Phenanthrol","1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphoethanolamine","Alanylphenylalanine","Ile-Leu","(2,2-dimethyl-3,4-dihydro-2H-chromen-6-yl)acetic acid","Octadecylphosphocholine","3-(2-methyl-3-oxo-2,5,6,7,8,8a-hexahydro-1H-naphthalen-1-yl)propanoic acid","4-Methylphthalic anhydride","Choline Alfoscerate","2-Propenoic acid, 3-[(4S,7R,7aR)-2,4,5,6,7,7a-hexahydro-3,7-dimethyl-1H-inden-4-yl]-2-methyl-, (2E)-","Spermine","2-Hydroxycinnamic acid, (2E)-","4,7-Dimethyl-1,3-benzothiazol-2-amine","Glu-Phe","L-Glutaminyl-L-isoleucine","1-Octadecanoyl-sn-glycero-3-phosphoethanolamine","4-Ethynylbiphenyl","Benzothiazole","Indole","Brefeldin A","1,7-Bis(4-hydroxyphenyl)heptan-3-one","Lovastatin","Bohlmann K2631","Phenethylamine","9-Fluorenone-2-carboxylic acid","Thr-Ile","Tert-butyl 4-methylbenzoate","1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine","Phe-Asp","Phe-Thr","8-[2-(4-Hydroxy-6-oxotetrahydro-2h-pyran-2-yl)ethyl]-7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2-methylbutanoate","N-(4-sulfamoylphenyl)propanamide","(Z)-11-hexadecenoic acid","(1S)-(+)-(10-Camphorsulfonyl)oxaziridine","gamma-Glutamylglutamate","Avocadene 2-acetate","Nadide Phosphate","(3aR,4R,5R,6aS)-5-hydroxy-4-[(3R)-3-hydroxy-5-phenylpentyl]-3,3a,4,5,6,6a-hexahydrocyclopenta[b]furan-2-one","7-hydroxy-5,8a-dimethyl-3-methylidene-2H,3H,3aH,5H,6H,7H,8H,8aH,9H,9aH-naphtho[2,3-b]furan-2-one","Xanthone","4-Methylbenzimidazole","Glu-Cys-Ala","3-(7-methyloctyl)-3,6-dihydro-1H-uro[3,4-c]uran-4-one","H-Leu-Glu-OH","2-(3-Phenylpropyl)Pyridine","Bis(2-ethylhexyl) phthalate","Povpc","1-O-Hexyl-2,3,5-trimethylhydroquinone","3,5-Dimethylpyridine","Stearic acid amide","1-(1-Ethynylcyclohexyl)piperidine","Linoleic Acid","3-prop-2-enoxy-N-(1,3-thiazol-2-yl)benzamide","Isobutyryl-L-carnitine","Curlone","Tyr-Leu","N-(2-Hydroxyethyl)heptadecanamide","PE(20:3(8Z,11Z,14Z)/0:0)","(10E,12Z)-octadeca-10,12-dienoic acid","12-Oxo-phytodienoic acid","3,5-Di-tert-butyl-4-hydroxybenzoic acid","1-Hexadecanoyl-sn-glycero-3-phosphoethanolamine","Asn-Pro-Lys","Cyclandelate","2-[3-(Formylamino)-2-hydroxybenzoylamino]-3-hydroxybutanoic acid methyl ester","Phe-Cys","Trp-Leu","(9E)-12-hydroperoxy-5,6-dihydroxy-4-methoxy-9,13,14-trimethyl-16-(2-methylpropyl)-17-azatricyclo[9.7.0.01,15]octadeca-9,13-diene-2,18-dione","Anthraquinone","1-(9Z-hexadecenoyl)-glycero-3-phosphoethanolamine","N-(2-cyanoethyl)-2-phenylethylamine","4-Acetamidobenzaldehyde","Sphingosine","17-Epiestriol","2,4-Dichlorobenzoic Acid","Elaidic Acid","N-(Tetradecanoyl)-sphing-4-enine","Ser-Phe","Trp-Pro-Lys","Lemidosul","N-isobutyl-2E,4Z-octadecadienoyl amine","2-[(4-Methoxyphenyl)sulfonyl]ethanamine","ANTIMYCIN A (A1 shown)","H-Phe-phe-OH","Myristoylcarnitine","Thr-Tyr","Methyl 3-[(2-methyl-1,3-thiazolidin-4-yl)formamido]propanoate","Isoquinolin-5-ol","Gentianine","3-(4-Sulfamoylphenyl)propanoic acid","4-(Methylthio)benzoic acid","Cadabicilone","Tris(2-chloroethyl) phosphate","Propionylcarnitine","Phosphoric Acid","3-((Dimethylamino)methyl)-2-methylindole","N,N-Dimethyldodecylamine","2-Hydroxy-4-methoxy-5-(2-methylbut-3-en-2-yl)benzaldehyde","2-Hydroxy-4,5',8a'-trimethyl-1'-oxo-4-vinyloctahydro-1'H-spiro[cyclopentane-1,2'-naphthalene]-5'-carboxylic acid","2-Monoolein","N-Butylaniline","Val-Tyr","Oridonin","11beta-Hydroxyandrostenedione","2-Ethylaniline","2,3-Xylidine","Muurolladie-3-one","13-keto-9Z,11E-octadecadienoic acid","Hexapropylene glycol","N,N-Diisopropylaniline","Compactin diol lactone","Diethylcarbamazine","Pentapropylene glycol","2-Palmitoylglycerol","Ser-Thr-Phe","Leu-Glu-Gly","Flunisolide","Citicoline","Ethylene dodecanedioate","4-Oxododecanedioic acid","Cysteine-glutathione disulfide","4-Methyl-1-phenylpentan-3-one","Cys-Gly-Glu","Adomet","Di(p-tolylidene)sorbitol","Gingerol","Stearidonic Acid","Val-Asp","4-(2,4,6-Trimethyl-3-cyclohexen-1-yl)-3-buten-2-one","L-Hexanoylcarnitine","Oleoyl Ethanolamide","Hexaethylene glycol","1-Pentadecanoyl-sn-glycero-3-phosphocholine","3-Amino-2-naphthoic acid","Dibenzylamine","4-Allyl-2,6-dimethoxyphenol","Tolazoline","Triphenylphosphine Oxide","Tenivastatin sodium","Trifluridine","Aldosterone","Lactarorufin A","16-Oxokahweol","Phe-Val","Guanosine Monophosphate","Indoline","Ubenimex","1-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1'-sn-glycerol)","L-Tyrosine 1,1-dimethylethyl ester","(S)-(-)-N,alpha-Dimethylbenzylamine","N-(2,4-Difluorophenyl)-1-{[4-(pyrrolidine-1-carbonyl)thiophen-2-YL]sulfonyl}piperidine-4-carboxamide","1-(1Z-hexadecenyl)-sn-glycero-3-phosphocholine","[(1S,7S,8S,8aS)-8-[2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl]-7-methyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-yl] (2S)-2-methylbutanoate","4-[(S)-amino(carboxy)methyl]-3-methylbenzoic acid","Laurolactam","Methionylglutamic acid","Simvastatin","Uridine-Diphosphate-N-Acetylglucosamine","Beta-Naphthoflavone","Erucamide","CID 790","4-Aminoethylbenzenesulfonyl fluoride hydrochloride","1-oxo-N-(4-sulfamoylphenyl)isochroman-3-carboxamide","Conjugated Linoleic Acid","Cadalene","Confertifolin","2,4,5-Trimethylaniline","Adenosine Phosphate","Ser-Leu","Lovastatin Hydroxy Acid","Diphenylcyclopropenone","Coronaric acid","Cyclomethicone 5","R-Deprenyl N-Oxide","DL-Leu-DL-Tyr","H-Glu-Tyr-OH","Phthalic anhydride","Met-His-Lys","1,3-Dicyclohexylurea","3,5-Di-tert-butylsalicylaldehyde","CID 11452543","N,N,N-trimethyl-2-(phosphonooxy)ethanaminium","Animicin A","2-Amino-1-phenylethanone","1-Heptadecanoyl-glycero-3-phosphoethanolamine","N-(oxolan-3-ylmethyl)-N-propylcyclohexanecarboxamide","Sphinganine","3-benzyl-2-(furan-2-yl)-3H-chromeno[2,3-d]pyrimidine-4,5-dione","Palmitic Acid","Acetyl-L-Carnitine","H-Leu-ile-OH","Triphenyl phosphate","1-Palmitoyl-sn-glycero-3-phosphocholine","Ser-Trp","N-(2,4-difluorophenyl)-2-(1,1-dioxo-2H-1lambda6,2,4-benzothiadiazin-3-yl)acetamide","N-Benzyl-4-methylbenzamide","Methyl 2-{[2-(4-methylphenyl)azepane-1-carbonyl]amino}acetate","Stearic Acid","14-Methylpentadecanoic acid","Microhelenin C","1-Methyladenosine","Dihydroactinidiolide, (+/-)-","LysoPC(18:3(9Z,12Z,15Z))","Oleoyl Ethyl Amide","3-Hydroxy-2-((9Z,12Z)-octadeca-9,12-dienoyloxy)propyl 2-(trimethylazaniumyl)ethyl phosphate","Ala-Gly-Glu","Gln-Lys-Leu","Pepstatin","Farnesyl acetone","Estrone","Pinolenic acid"],"pubmed_abstract":["INTRODUCTION: Untargeted metabolomics is a popular method by which researchers measure a large portion of the metabolites present in a biological system at once. This approach usually results in complex data sets containing tens to hundreds of thousands of observations which require sophisticated data analysis workflows. To help with the functional interpretation of the data, researchers often rely on enrichment analysis. However, little advice is available on what method to use, and, to the best of our knowledge, there is no comparison of popular approaches available for in vitro data with a focus on toxicological and pharmacological testing. OBJECTIVES: In this study, we compared three popular enrichment analysis approaches—Metabolite Set Enrichment Analysis (MSEA), Mummichog and Over Representation Analysis (ORA)—with data obtained by treating Hep-G2 cells with 11 compounds with five different mechanisms of action. We compared the results and assessed the consistency of the individual methods as well as their correctness. METHODS: Hep-G2 cells were treated with subtoxic concentrations of 11 test compounds. After preparation, samples were measured on an Elute UHPLC coupled to a timsTOF Pro (both Bruker). Spectra were processed in MetaboScape (Bruker) and annotated using spectral library search. Datasets were further processed using R and enrichment analysis was performed in MetaboAnalyst. RESULTS: Overall, we observed a low to moderate similarity between different enrichment methods with the highest similarity between MSEA and Mummichog. Further, Mummichog outperformed both MSEA and ORA in terms of consistency and correctness. CONCLUSION: In our comparison, Mummichog showed the best performance for in vitro untargeted metabolomics data."],"pubmed_title":["Comparison of popular enrichment methods for untargeted in vitro metabolomics."],"pubmed_authors":["Schermer Yannik Y, Wagner Frederic F, Stegmüller Simone S, Richling Elke E"],"additional_accession":[]},"is_claimable":false,"name":"Comparison of popular enrichment methods for untargeted in vitro metabolomics.","description":"Untargeted metabolomics is a popular method by which researchers measure a large portion of the metabolites present in a biological system at once. This approach usually results in complex data sets containing tens to hundreds of thousands of observations which require sophisticated data analysis workflows. To help with the functional interpretation of the data, researchers often rely on enrichment analysis. However, little advice is available on what method to use, and, to the best of our knowledge, there is no comparison of popular approaches available for in vitro data with a focus on toxicological and pharmacological testing.
In this study, we compared three popular enrichment analysis approaches-Metabolite Set Enrichment Analysis (MSEA), Mummichog and Over Representation Analysis (ORA)-with data obtained by treating Hep-G2 cells with 11 compounds with five different mechanisms of action. We compared the results and assessed the consistency of the individual methods as well as their correctness.
Hep-G2 cells were treated with subtoxic concentrations of 11 test compounds. After preparation, samples were measured on an Elute UHPLC coupled to a timsTOF Pro (both Bruker). Spectra were processed in MetaboScape (Bruker) and annotated using spectral library search. Datasets were further processed using R and enrichment analysis was performed in MetaboAnalyst.
Overall, we observed a low to moderate similarity between different enrichment methods with the highest similarity between MSEA and Mummichog. Further, Mummichog outperformed both MSEA and ORA in terms of consistency and correctness.
In our comparison, Mummichog showed the best performance for in vitro untargeted metabolomics data.
","dates":{"publication":"2025-08-11","submission":"2025-03-19"},"accession":"MTBLS12332","cross_references":{"pubmed":["40715905"],"PubChem":["CID 24066906","CID 616","CID 92970719","CID 156373480","CID 12227668","CID 109265","CID 11305","CID 9547071","CID 9547099","CID 9547069","CID 46907872","CID 9547068","CID 132285229","CID 9547070","CID 5356421","CID 440043","CID 135398597","CID 16061101","CID 11868","CID 246868","CID 75528849","CID 22467566","CID 9378","CID 10443535","CID 818219","CID 23789044","CID 3634442","CID 12575964","CID 70318","CID 688023","CID 102571644","CID 2733480","CID 156602850","CID 5283005","CID 16727166","CID 4765","CID 6131","CID 315878","CID 1899","CID 15868","CID 45783154","CID 138052","CID 16061050","CID 5702199","CID 19266","CID 110491","CID 904","CID 6083","CID 6022","CID 447145","CID 2000","CID 96814","CID 18218182","CID 2266","CID 77278","CID 30647","CID 13625","CID 171548","CID 7009597","CID 7010531","CID 7010579","CID 790","CID 3330","CID 311","CID 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