An in-house MS2 spectral library containing experimental MS2 spectra and retention times (RT) for 391 compounds obtained from standards[1][2] was used for annotation of detected features using 3 criteria: (i) accurate mass (AM) match (tolerance: 0.01 Da), (ii) RT match (tolerance: 1 min), and (iii) MS2 spectrum match (similarity >70%). The MS2 similarity was scored by the simple dot product without any weighting (at least 2 MS2 peaks match with the reference spectra). The MS2 similarities with reference spectra were matched to any of the CorrDec[3] or the MS2Dec[4] deconvoluted MS2 spectra of the 3 collision energies (0, 10, and 30 eV). We used peak areas of the representative ion (usually [M+H]+) for the relative quantification of metabolites.
Ref:
[1] Naz S, Gallart-Ayala H, Reinke SN, Mathon C, Blankley R, Chaleckis R, Wheelock CE. Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition. Anal Chem. 2017 Aug 1;89(15):7933-7942. doi:10.1021/acs.analchem.7b00925. PMID:28641411.
[2] Tada I, Tsugawa H, Meister I, Zhang P, Shu R, Katsumi R, Wheelock CE, Arita M, Chaleckis R. Creating a Reliable Mass Spectral-Retention Time Library for All Ion Fragmentation-Based Metabolomics. Metabolites. 2019 Oct 26;9(11):251. doi:10.3390/metabo9110251. PMID:31717785.
[3] Tada I, Chaleckis R, Tsugawa H, Meister I, Zhang P, Lazarinis N, Dahlén B, Wheelock CE, Arita M. Correlation-Based Deconvolution (CorrDec) To Generate High-Quality MS2 Spectra from Data-Independent Acquisition in Multisample Studies. Anal Chem. 2020 Aug 18;92(16):11310-11317. doi:10.1021/acs.analchem.0c01980. PMID:32648737.
[4] Tsugawa H, Cajka T, Kind T, Ma Y, Higgins B, Ikeda K, Kanazawa M, VanderGheynst J, Fiehn O, Arita M. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nat Methods. 2015 Jun;12(6):523-6. doi:10.1038/nmeth.3393. PMID:25938372.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - Positive (LC-MS (Positive))"],"chromatography_protocol":["Injection volume was 1 μl (injection sequence see Table S2 in the paper associated with this study). The LC-MS method was described in previous publications[1]-[3]. Briefly, metabolite separation was performed on an Agilent 1290 Infinity II system using SeQuant ZIC-HILIC (Merck, Darmstadt, Germany) column. Sample analysis was performed using water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B). The elution gradient used was as follows: isocratic step at 95% B for 1.5 min, 95% B to 40% B in 12 min and maintained at 40% B for 2 min. The flow rate was 0.4 ml/min.
Ref:
[1] Naz S, Gallart-Ayala H, Reinke SN, Mathon C, Blankley R, Chaleckis R, Wheelock CE. Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition. Anal Chem. 2017 Aug 1;89(15):7933-7942. doi:10.1021/acs.analchem.7b00925. PMID:28641411.
[2] Chaleckis R, Naz S, Meister I, Wheelock CE. LC-MS-Based Metabolomics of Biofluids Using All-Ion Fragmentation (AIF) Acquisition. Methods Mol Biol. 2018;1730:45-58. doi:10.1007/978-1-4939-7592-1_3. PMID:29363064.
[3] Tada I, Tsugawa H, Meister I, Zhang P, Shu R, Katsumi R, Wheelock CE, Arita M, Chaleckis R. Creating a Reliable Mass Spectral-Retention Time Library for All Ion Fragmentation-Based Metabolomics. Metabolites. 2019 Oct 26;9(11):251. doi:10.3390/metabo9110251. PMID:31717785.
"],"publication":["Untargeted LC-MS metabolomics for the analysis of micro-scaled extracellular metabolites from hepatocytes. 10.2116/alsci.20n032. PMID:33342928"],"submitter_affiliation":["Nagoya City University"],"submitter_name":["Romanas Chaleckis"],"organism_part":["Hep-G2 cell","mixture","water"],"technology_type":["mass spectrometry"],"disease":[""],"extraction_protocol":["CCM samples were thawed at room temperature and 1 μl of each sample was extracted using 50, 100 μl or 200 μl acetonitrile containing 5 technical internal standards (tIS), see Table S1 in the paper associated with this study. Then samples were centrifuged at 4 °C for 15 min at 20000 x g. 40 μl of the supernatant were transferred to a 96-well 0.2 ml PCR plate PCR-96-MJ (BMBio, Tokyo, Japan). The plate was sealed with a pierceable seal (4titude, Wotton, UK) for 3 s at 180 °C using a plate sealer (BioRad PX-1, CA, USA) and kept at 4 °C during the LC-MS measurement.
"],"organism":["blank","Reference compound","Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS1794"],"author":["Ken-Ichiro Kamei. Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan.","Rodi Abdalkader. Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan.","Isabel Meister. Gunma University Initiative for Advanced Research (GIAR), Gunma University, Gunma, Japan and Division of Physiological Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.","Pei Zhang. Gunma University Initiative for Advanced Research (GIAR), Gunma University, Gunma, Japan and Division of Physiological Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.","Romanas Chaleckis. Gunma University Initiative for Advanced Research (GIAR), Gunma University, Gunma, Japan and Division of Physiological Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. romanas.chaleckis@gmail.com.","Craig Wheelock. Gunma University Initiative for Advanced Research (GIAR), Gunma University, Gunma, Japan and Division of Physiological Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden."],"data_transformation_protocol":["Data was transformed from Agilent .d format to .abf format using Reifycs Abf (Analysis Base File) Converter (https://www.reifycs.com/AbfConverter/). Data were processed in MS-DIAL version 4.20[1] (detailed parameters in Tables S3 and S4 in the publication associated with this study).
Ref:
[1] Tsugawa H, Cajka T, Kind T, Ma Y, Higgins B, Ikeda K, Kanazawa M, VanderGheynst J, Fiehn O, Arita M. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nat Methods. 2015 Jun;12(6):523-6. doi:10.1038/nmeth.3393. PMID:25938372.
"],"study_factor":["Extraction volume","Replicate","Timepoint"],"submitter_email":["romanas.chaleckis@gmail.com"],"sample_collection_protocol":["HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 10% (v/v) FBS, 1% penicillin/streptomycin, and 1 mM nonessential amino acids. The cells were passaged with trypsin-EDTA solutions at a 1:10 to 1:20 subculture ratio. For the collection of extracellular metabolites 3 x 10^5 cells were seeded into T-25 flasks in a final volume of 15 ml of the CCM. Cells were then incubated at 37 °C and 5% CO2. At 3 timepoints (0 - control, 24 and 72 h) 1 ml of extracellular CCM was taken and preserved at -80 °C.
"],"omics_type":["Metabolomics"],"study_design":["Micro-scale cell culture medium","tandem mass spectrometry","liquid chromatography-mass spectrometry","untargeted metabolites"],"curator_keywords":["Micro-scale cell culture medium","tandem mass spectrometry","liquid chromatography-mass spectrometry","untargeted metabolites"],"mass_spectrometry_protocol":["Data was acquired on an Agilent 6550 Q-TOF-MS system with a mass range of 40−1200 m/z in positive ionization all ion fragmentation mode (AIF) including 3 sequential experiments at alternating collision energies: 1 full scan at 0 eV, followed by 1 MS/MS scan at 10 eV, and then followed by 1 MS/MS scan at 30 eV. The data acquisition rate was 6 scans/s.
"],"metabolite_name":["Taurine","Riboflavin","Creatine","N-Acetyl-D-galactosamine","Betaine","Butyryl-carnitine","Methionine sulfone","Cytosine","N1-Methyladenosine","Tyrosine","Threonine","Methionine","Cytidine","Carnitine","Methylguanosine","Valeryl-carnitine","Hypoxanthine","Homostachydrine","Nicotinamide","N2-Dimethylguanosine","4-Acetamidobutyric acid","Methylhistidine","4-Hydroxyproline","Hexanoyl-carnitine","Trimethylamine N-oxide","Hippuric acid","Methionine sulfoxide","Pyrantel","5-Fluorocytosine","Proline","Leucine","Decanoyl-carnitine","CHES","Penicillin G","Quinaldic acid","Indoleacetic acid","Pyroglutamic acid","Kynurenine","Thiamine","Isoleucine","Creatinine","Adenosine","Phenylalanine","Propionyl-carnitine","Tryptophan","Xanthosine","Phenylacetylglycine","Pyridoxine","Pseudouridine","Xanthine","PIPES","7-Methylguanine","Valine","Proline betaine","Acetylcarnitine","Choline","HEPES","Sarcosine","Dimethylarginine","N-Methyl-proline","Glutamine","Pantothenic acid"],"pubmed_abstract":["Metabolome analysis in micro physiological models is a challenge due to the low volume of the cell culture medium (CCM). Here, we report a LC-MS-based untargeted metabolomics protocol for the detection of hepatocyte extracellular metabolites from micro-scale samples of CCM. Using a single LC-MS method we have detected 57 metabolites of which 27 showed >2-fold shifts after 72-hour incubation. We demonstrate that micro-scale CCM samples can be used for modelling micro-physiological temporal dynamics in metabolite intensities."],"pubmed_title":["Untargeted LC-MS Metabolomics for the Analysis of Micro-scaled Extracellular Metabolites from Hepatocytes."],"pubmed_authors":["Abdalkader Rodi R, Chaleckis Romanas R, Meister Isabel I, Zhang Pei P, Wheelock Craig E CE, Kamei Ken-Ichiro KI"],"description_synonyms":["small volume, scale tissue, Culture Techniques, familial cavernous angioma, Procedures, determination, cerebral capillary malformations, liver parenchymal cell, Ass-1, peltate hair, familial, cerebral cavernous malformations, Metabonomic, secondary metabolites, Hepatocyte, Metabonomics, Profiles, Progress Reports, Procedure, Cell, temporal, low volume, Hepatic Cells, method, Techniques, primary metabolites, Investigative, Investigative Report, cavernous angioma, Hepatic Cell, Method, Summary Report, method used in an experiment, AA408052, chemical analysis, Research Reports, Studies, fold, parenchymal liver cell, scales, Metabolomic, metabolites., Cell Culture, Metabolic Profile, Summary Reports, Technique, cavernous malformations of CNS and retina, Metabolic Profiles, Culture Technique, scale, Metabolic, Progress Report, Metabolomes, parenchymal hepatic cell, Profile, plant peltate hair, Field, metabolite, Methodological, study protocol, Methodological Study, hyperkeratotic cutaneous capillary-Venous malformations associated with cerebral capillary malformations, Cell Culture Technique, plan specification, Study, ASS, Progress, extracellular, Report, Field Reports, 10^[-6], cavernous angiomatous malformations, Methodological Studies, Reports, CCM, Cells, Cell Cultures, Hepatic, assay, Investigative Reports, Summary, Field Report, incidence, cerebral cavernous malformations 1"],"pubmed_title_synonyms":["extracellular, Hepatic Cells, 10^[-6], determination, chemical analysis, Cells, Metabonomic, Hepatic, Hepatocyte, assay, Metabonomics, Metabolomic, Cell, Hepatic Cell."],"pubmed_abstract_synonyms":["small volume, scale tissue, Culture Techniques, Procedures, determination, liver parenchymal cell, Ass-1, peltate hair, Metabonomic, secondary metabolites, Hepatocyte, Metabonomics, Profiles, Progress Reports, Procedure, Cell, temporal, low volume, Hepatic Cells, method, Techniques, primary metabolites, Investigative, Investigative Report, Hepatic Cell, Method, Summary Report, method used in an experiment, AA408052, chemical analysis, Research Reports, Studies, fold, parenchymal liver cell, scales, Metabolomic, metabolites., Cell Culture, Metabolic Profile, Summary Reports, Technique, Metabolic Profiles, Culture Technique, scale, Metabolic, Progress Report, Metabolomes, parenchymal hepatic cell, Profile, plant peltate hair, Field, metabolite, Methodological, study protocol, Methodological Study, Cell Culture Technique, plan specification, Study, ASS, Progress, extracellular, Report, Field Reports, 10^[-6], Methodological Studies, h, Reports, Cells, Cell Cultures, Hepatic, assay, Investigative Reports, Summary, Field Report, incidence"],"name_synonyms":["extracellular, Hepatic Cells, 10^[-6], determination, chemical analysis, Cells, Metabonomic, Hepatic, Hepatocyte, assay, Metabonomics, Metabolomic, Cell, Hepatic Cell."],"additional_accession":[]},"is_claimable":false,"name":"Untargeted LC-MS metabolomics for the analysis of micro-scaled extracellular metabolites from hepatocytes","description":"Metabolome analysis in micro physiological models is a challenge due to the low volume of cell culture medium (CCM). Here, we report a LC-MS-based untargeted metabolomics protocol for the detection of hepatocyte extracellular metabolites from micro-scale samples of CCM. Using a single LC-MS method we have detected 57 metabolites of which 27 showed >2-fold shifts after 72-hours incubation. We demonstrate that micro-scale CCM samples can be used for modelling micro-physiological temporal dynamics in metabolite intensities.","dates":{"publication":"2021-01-04","submission":"2020-06-12"},"accession":"MTBLS1794","cross_references":{"MetaboLights":["MTBLC18208","MTBLC27480","MTBLC18089","MTBLC18386","MTBLC16411","MTBLC17645","MTBLC7916","MTBLC68830","MTBLC70749","MTBLC18183","MTBLC86050","MTBLC17154","MTBLC7676","MTBLC28867","MTBLC73024","MTBLC17015","MTBLC15318","MTBLC17368","MTBLC19289","MTBLC18107","MTBLC25307","MTBLC17802","MTBLC28037","MTBLC28044","MTBLC25017","MTBLC15724","MTBLC28683","MTBLC16335","MTBLC24898","MTBLC27897","MTBLC5757","MTBLC16811","MTBLC16709","MTBLC2274","MTBLC27266","MTBLC35280","MTBLC90344","MTBLC15354","MTBLC17750","MTBLC18186","MTBLC17126","MTBLC26271","MTBLC16919","MTBLC16737","MTBLC15891","MTBLC132188","MTBLC20392","MTBLC15611","MTBLC16020","MTBLC16040","MTBLC26986","MTBLC49033","MTBLC28300","MTBLC17562","MTBLC86468","MTBLC137682","MTBLC26948","MTBLC8654","MTBLC44302","MTBLC5100","MTBLC39033","MTBLC46756"],"pubmed":["33342928"],"ChEBI":["CHEBI:18208","CHEBI:27480","CHEBI:18089","CHEBI:18386","CHEBI:16411","CHEBI:17645","CHEBI:7916","CHEBI:68830","CHEBI:70749","CHEBI:18183","CHEBI:86050","CHEBI:17154","CHEBI:7676","CHEBI:28867","CHEBI:73024","CHEBI:17015","CHEBI:15318","CHEBI:17368","CHEBI:19289","CHEBI:18107","CHEBI:25307","CHEBI:17802","CHEBI:28037","CHEBI:28044","CHEBI:25017","CHEBI:15724","CHEBI:28683","CHEBI:16335","CHEBI:24898","CHEBI:27897","CHEBI:5757","CHEBI:16811","CHEBI:16709","CHEBI:2274","CHEBI:27266","CHEBI:35280","CHEBI:90344","CHEBI:15354","CHEBI:17750","CHEBI:18186","CHEBI:17126","CHEBI:26271","CHEBI:16919","CHEBI:16737","CHEBI:15891","CHEBI:132188","CHEBI:20392","CHEBI:15611","CHEBI:16020","CHEBI:16040","CHEBI:26986","CHEBI:49033","CHEBI:28300","CHEBI:17562","CHEBI:86468","CHEBI:137682","CHEBI:26948","CHEBI:8654","CHEBI:44302","CHEBI:5100","CHEBI:39033","CHEBI:46756"]}}