MTBLS127: Resveratrol metabolism in HepG2 (human hepatocytes), HaCaT (human keratinocytes), and C2C12 (mouse myoblasts)

Abstract

Resveratrol (RESV) is a plant polyphenol, which is thought to have beneficial metabolic effects in laboratory animals as well as in humans. Following oral administration, RESV is immediately catabolized, resulting in low bioavailability. This study compared RESV metabolites and their tissue distribution after oral uptake and skin absorption. Metabolomic analysis of various mouse tissues revealed that RESV can be absorbed and metabolized through skin. We detected sulfated and glucuronidated RESV metabolites, as well as dihydroresveratrol. These metabolites are thought to have lower pharmacological activity than RESV. Similar quantities of most RESV metabolites were observed 4 h after oral or skin administration, except that glucuronidated RESV metabolites were more abundant in skin after topical RESV application than after oral administration. This result is consistent with our finding of glucuronidated RESV metabolites in cultured skin cells. RESV applied to mouse ears significantly suppressed inflammation in the TPA inflammation model. The skin absorption route could be a complementary, potent way to achieve therapeutic effects with RESV.

Cells were treated with 0, 20 or 200 µM RESV for 4 h. After washing with PBS, cells were lysed, and metabolites were extracted and analyzed by LC-MS. Peak areas metabolites were normalized by peak areas of spiked internal standards (10 nmol 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES). 3 samples for each condition (except for HaCaT 200 µM, 2 samples).

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 Authors: Itsuo Murakami, Romanas Chaleckis, Tomáš Pluskal, Ken Ito, Kousuke Hori, Masahiro Ebe, Mitsuhiro Yanagida, Hiroshi Kondoh

  Release date: 15-Dec-2014

 Status: Public

Organism(s)

Mus musculus

Homo sapiens

  Study Design

untargeted metabolites

NCIT:Resveratrol

CHMO:tandem mass spectrometry

CHMO:high-performance liquid chromatography-mass spectrometry

EFO:cell line

  Experimental Factors

Cell line

Resveratrol concentration

Protocol Description
Sample collection Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 1% penicillin/streptomycin and 2 mM L-glutamine with some modifications as follows; DMEM (1.0 g/l glucose) with 10% fetal calf serum (FCS) for HepG2; DMEM (4.5 g/l glucose) with 10% FCS for HaCaT; DMEM (4.5 g/l glucose) with 20% FCS for C2C12. C2C12 cells were differentiated into myotubes by switching medium into DMEM containing 2% horse serum. Cells were plated on a 10 cm dish and allowed to adhere for 18 h. Cells were treated with 20 or 200 µM RESV. After 4 h incubation, cells were washed with PBS twice, followed by addition of 2 ml 50% ice-cold methanol. Collected cells were transferred into tubes on ice for further processing.
Extraction After addition of internal standards (10 nmol HEPES and PIPES) to metabolome samples and brief vortexing, samples were transferred to Amicon Ultra 10 kDa cut-off filters (Millipore, Billerica, MA, USA) to remove proteins. Following sample concentration by vacuum evaporation, each sample was resuspended in 40 µl 50% acetonitrile and 1 µl was used for liquid chromatography-mass spectrometry (LC-MS) analysis.
Chromatography LC-MS data were obtained using a Paradigm MS4 HPLC system (Michrom Bioresources, Auburn, CA, USA) coupled to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), as described previously [1]. Briefly, metabolites were separated on a ZIC-pHILIC column (Merck SeQuant, Sweden; 150 x 2.1 mm, 5 µm particle size) using acetonitrile (A) and 10 mM ammonium carbonate buffer, pH 9.3 (B) as the mobile phase, with gradient elution from 80% A to 20% A in 30 min, at a flow rate of 100 µl/min. Sample injection volume was 1 µl per run.

Ref:
[1] Pluskal T, Nakamura T, Villar-Briones A, Yanagida M (2010) Metabolic profiling of the fission yeast S. pombe: quantification of compounds under different temperatures and genetic perturbation. Mol Biosyst 6:182–198. doi: 10.1039/b908784b PMID:20024080
Mass spectrometry The LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was operated in full scan mode with the scan range of 100-1,000 m/z. Each sample was analyzed twice, once in negative and once in positive ionization mode.
Data transformation Peak areas of metabolites of interest, within the raw LC-MS data, were measured using the MZmine 2.10 software and normalized using the peak areas of HEPES and PIPES. The complete workflow and processing parameters are summarized in the publication.
Metabolite identification Metabolites were identified by comparing their m/z values and retention times with authentic pure standards. If no standard was available, metabolites were assigned by analysis of MS/MS spectra (MS/MS), as described in the publication. In cases where no standard compound or MS/MS spectrum was available, compounds were identified by generating chemical formulae using m/z values in MZmine 2.10 module and given MS status.
Source Name Organism Organism part Protocol REF Sample Name Cell line Resveratrol concentration Unit
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 1 RESV 0uM HepG2 0 micromole
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 2 RESV 0uM HepG2 0 micromole
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 3 RESV 0uM HepG2 0 micromole
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 1 RESV 20uM HepG2 20 micromole
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 2 RESV 20uM HepG2 20 micromole
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 3 RESV 20uM HepG2 20 micromole
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 1 RESV 200uM HepG2 200 micromole
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 2 RESV 200uM HepG2 200 micromole
HepG2 Homo sapiens Hep-G2 cell Sample collection HepG2 3 RESV 200uM HepG2 200 micromole
HaCaT Homo sapiens HaCaT cell Sample collection HaCaT 1 RESV 0uM HaCaT 0 micromole
HaCaT Homo sapiens HaCaT cell Sample collection HaCaT 2 RESV 0uM HaCaT 0 micromole
HaCaT Homo sapiens HaCaT cell Sample collection HaCaT 3 RESV 0uM HaCaT 0 micromole
HaCaT Homo sapiens HaCaT cell Sample collection HaCaT 1 RESV 20uM HaCaT 20 micromole
HaCaT Homo sapiens HaCaT cell Sample collection HaCaT 2 RESV 20uM HaCaT 20 micromole
HaCaT Homo sapiens HaCaT cell Sample collection HaCaT 3 RESV 20uM HaCaT 20 micromole
HaCaT Homo sapiens HaCaT cell Sample collection HaCaT 1 RESV 200uM HaCaT 200 micromole
HaCaT Homo sapiens HaCaT cell Sample collection HaCaT 2 RESV 200uM HaCaT 200 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 1 RESV 0uM C2C12 0 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 2 RESV 0uM C2C12 0 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 3 RESV 0uM C2C12 0 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 1 RESV 20uM C2C12 20 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 2 RESV 20uM C2C12 20 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 3 RESV 20uM C2C12 20 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 1 RESV 200uM C2C12 200 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 2 RESV 200uM C2C12 200 micromole
C2C12 Mus musculus C2C12 cell Sample collection C2C12 3 RESV 200uM C2C12 200 micromole

Assay  1

Assay file name: a_mtblsxxx2_metabolite_profiling_mass_spectrometry-1.txt
Technology: mass spectrometry
Platform: LTQ Orbitrap Classic (Thermo Scientific)

Instrumentation

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
HepG2 1 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 1 RESV 0uM POS HepG2 1 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HepG2 2 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 2 RESV 0uM POS HepG2 2 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HepG2 3 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 3 RESV 0uM POS HepG2 3 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HepG2 1 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 1 RESV 20uM POS HepG2 1 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HepG2 2 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 2 RESV 20uM POS HepG2 2 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HepG2 3 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 3 RESV 20uM POS HepG2 3 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HepG2 1 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 1 RESV 200uM POS HepG2 1 RESV 200uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HepG2 2 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 2 RESV 200uM POS HepG2 2 RESV 200uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HepG2 3 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 3 RESV 200uM POS HepG2 3 RESV 200uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HaCaT 1 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 1 RESV 0uM POS HaCaT 1 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HaCaT 2 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 2 RESV 0uM POS HaCaT 2 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HaCaT 3 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 3 RESV 0uM POS HaCaT 3 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HaCaT 1 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 1 RESV 20uM POS HaCaT 1 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HaCaT 2 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 2 RESV 20uM POS HaCaT 2 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HaCaT 3 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 3 RESV 20uM POS HaCaT 3 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HaCaT 1 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 1 RESV 200uM POS HaCaT 1 RESV 200uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
HaCaT 2 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 2 RESV 200uM POS HaCaT 2 RESV 200uM POS.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv

Assay  2

Assay file name: a_mtbls127_metabolite_profiling_mass_spectrometry-1.txt
Technology: mass spectrometry
Platform: LTQ Orbitrap Classic (Thermo Scientific)

Instrumentation

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
C2C12 1 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 1 RESV 0uM NEG C2C12 1 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
C2C12 2 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 2 RESV 0uM NEG C2C12 2 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
C2C12 3 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 3 RESV 0uM NEG C2C12 3 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
C2C12 1 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 1 RESV 20uM NEG C2C12 1 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
C2C12 2 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 2 RESV 20uM NEG C2C12 2 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
C2C12 3 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 3 RESV 20uM NEG C2C12 3 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
C2C12 1 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 1 RESV 200uM NEG C2C12 1 RESV 200uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
C2C12 2 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 2 RESV 200uM NEG C2C12 2 RESV 200uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
C2C12 3 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 3 RESV 200uM NEG C2C12 3 RESV 200uM NEG.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv

Assay  3

Assay file name: a_mtblsxxx2_metabolite_profiling_mass_spectrometry.txt
Technology: mass spectrometry
Platform: LTQ Orbitrap Classic (Thermo Scientific)

Instrumentation

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
HepG2 1 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 1 RESV 0uM NEG HepG2 1 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 2 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 2 RESV 0uM NEG HepG2 2 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 3 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 3 RESV 0uM NEG HepG2 3 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 1 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 1 RESV 20uM NEG HepG2 1 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 2 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 2 RESV 20uM NEG HepG2 2 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 3 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 3 RESV 20uM NEG HepG2 3 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 1 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 1 RESV 200uM NEG HepG2 1 RESV 200uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 2 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 2 RESV 200uM NEG HepG2 2 RESV 200uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 3 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HepG2 3 RESV 200uM NEG HepG2 3 RESV 200uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HaCaT 1 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 1 RESV 0uM NEG HaCaT 1 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HaCaT 2 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 2 RESV 0uM NEG HaCaT 2 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HaCaT 3 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 3 RESV 0uM NEG HaCaT 3 RESV 0uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HaCaT 1 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 1 RESV 20uM NEG HaCaT 1 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HaCaT 2 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 2 RESV 20uM NEG HaCaT 2 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HaCaT 3 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 3 RESV 20uM NEG HaCaT 3 RESV 20uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HaCaT 1 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 1 RESV 200uM NEG HaCaT 1 RESV 200uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HaCaT 2 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry negative 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap HaCaT 2 RESV 200uM NEG HaCaT 2 RESV 200uM NEG.mzML Data transformation Metabolite identification m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv

Assay  4

Assay file name: a_mtbls127_metabolite_profiling_mass_spectrometry.txt
Technology: mass spectrometry
Platform: LTQ Orbitrap Classic (Thermo Scientific)

Instrumentation

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
C2C12 1 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 1 RESV 0uM POS C2C12 1 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
C2C12 2 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 2 RESV 0uM POS C2C12 2 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
C2C12 3 RESV 0uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 3 RESV 0uM POS C2C12 3 RESV 0uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
C2C12 1 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 1 RESV 20uM POS C2C12 1 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
C2C12 2 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 2 RESV 20uM POS C2C12 2 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
C2C12 3 RESV 20uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 3 RESV 20uM POS C2C12 3 RESV 20uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
C2C12 1 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 1 RESV 200uM POS C2C12 1 RESV 200uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
C2C12 2 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 2 RESV 200uM POS C2C12 2 RESV 200uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
C2C12 3 RESV 200uM Extraction Chromatography Michrom BioResources Paradigm MS4 HPLC system ZIC-pHILIC (5 µm, 2.1 mm × 150 mm; Merck Sequant) HILIC Mass spectrometry positive 100-1000 Thermo Scientific LTQ Orbitrap MS:electrospray ionization orbitrap C2C12 3 RESV 200uM POS C2C12 3 RESV 200uM POS.mzML Data transformation Metabolite identification m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
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List of study files   Subset

File
C2C12 1 RESV 0uM NEG.mzML
C2C12 1 RESV 0uM POS.mzML
metexplore_mapping.json
C2C12 1 RESV 200uM NEG.mzML
C2C12 1 RESV 20uM NEG.mzML
C2C12 3 RESV 0uM NEG.mzML
C2C12 2 RESV 200uM NEG.mzML
C2C12 2 RESV 0uM NEG.mzML
C2C12 1 RESV 20uM POS.mzML
C2C12 2 RESV 0uM POS.mzML
C2C12 2 RESV 20uM POS.mzML
C2C12 2 RESV 200uM POS.mzML
C2C12 1 RESV 200uM POS.mzML
C2C12 2 RESV 20uM NEG.mzML
HaCaT 1 RESV 0uM POS.mzML
C2C12 3 RESV 200uM NEG.mzML
C2C12 3 RESV 20uM POS.mzML
HaCaT 1 RESV 0uM NEG.mzML
HaCaT 1 RESV 20uM NEG.mzML
C2C12 3 RESV 200uM POS.mzML
HaCaT 1 RESV 200uM POS.mzML
HaCaT 1 RESV 20uM POS.mzML
C2C12 3 RESV 20uM NEG.mzML
C2C12 3 RESV 0uM POS.mzML
HaCaT 1 RESV 200uM NEG.mzML
HepG2 1 RESV 0uM NEG.mzML
HaCaT 2 RESV 200uM NEG.mzML
audit
HaCaT 2 RESV 20uM POS.mzML
HaCaT 2 RESV 0uM NEG.mzML
HaCaT 3 RESV 0uM NEG.mzML
HaCaT 3 RESV 20uM POS.mzML
HaCaT 2 RESV 20uM NEG.mzML
HaCaT 2 RESV 0uM POS.mzML
HaCaT 2 RESV 200uM POS.mzML
HaCaT 3 RESV 0uM POS.mzML
HaCaT 3 RESV 20uM NEG.mzML
HepG2 1 RESV 20uM NEG.mzML
HepG2 1 RESV 200uM POS.mzML
HepG2 1 RESV 20uM POS.mzML
HepG2 2 RESV 200uM NEG.mzML
HepG2 2 RESV 200uM POS.mzML
HepG2 1 RESV 0uM POS.mzML
HepG2 1 RESV 200uM NEG.mzML
HepG2 2 RESV 0uM NEG.mzML
HepG2 2 RESV 20uM NEG.mzML
HepG2 2 RESV 20uM POS.mzML
HepG2 2 RESV 0uM POS.mzML
HepG2 3 RESV 0uM NEG.mzML
HepG2 3 RESV 0uM POS.mzML
a_mtblsxxx2_metabolite_profiling_mass_spectrometry-1.txt
HepG2 3 RESV 200uM NEG.mzML
HepG2 3 RESV 200uM POS.mzML
i_Investigation.txt
a_mtbls127_metabolite_profiling_mass_spectrometry.txt
s_MTBLSXXX2.txt
HepG2 3 RESV 20uM NEG.mzML
a_mtblsxxx2_metabolite_profiling_mass_spectrometry.txt
m_mtbls127_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
m_mtbls127_metabolite_profiling_mass_spectrometry_v2_maf.tsv
HepG2 3 RESV 20uM POS.mzML
m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_mafXX.tsv
m_mtblsxxx2_metabolite_profiling_mass_spectrometry-1_v2_maf.tsv
m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_maf.tsv
m_mtblsxxx2_metabolite_profiling_mass_spectrometry_v2_mafXX.tsv
a_mtbls127_metabolite_profiling_mass_spectrometry-1.txt

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Validations marked with (*) have been allowed by the MetaboLights Curators.
Click here for the detailed description of Validations.
Condition Status Description Requirement Group Message
PASSES Study Title MANDATORY STUDY OK
PASSES Study Description MANDATORY STUDY OK
PASSES Study text successfully parsed OPTIONAL STUDY OK
PASSES Study Contact(s) have listed email MANDATORY CONTACT OK
PASSES Sample(s) MANDATORY SAMPLES OK
PASSES Sample Name consistency check MANDATORY ASSAYS OK
PASSES Publication(s) associated with this Study MANDATORY PUBLICATION OK
PASSES Minimal Experimental protocol MANDATORY PROTOCOLS OK
PASSES Comprehensive Experimental protocol OPTIONAL PROTOCOLS OK
PASSES Extraction protocol description MANDATORY PROTOCOLS OK
PASSES Data transformation protocol description MANDATORY PROTOCOLS OK
PASSES Metabolite Identification protocol description MANDATORY PROTOCOLS OK
PASSES Mass spectrometry protocol description MANDATORY PROTOCOLS OK
PASSES Chromatography protocol description MANDATORY PROTOCOLS OK
PASSES Sample Collection protocol description MANDATORY PROTOCOLS OK
PASSES Protocols text successfully parsed OPTIONAL PROTOCOLS OK
PASSES Organism name MANDATORY ORGANISM OK
PASSES Organism part MANDATORY ORGANISM OK
PASSES Study Factors MANDATORY FACTORS OK
PASSES Assay platform information OPTIONAL ASSAYS OK
PASSES Assay has raw files referenced MANDATORY FILES OK
PASSES Assay referenced raw files detection in filesystem MANDATORY FILES OK
PASSES Raw files in the Assay(s) have the correct format MANDATORY FILES OK
PASSES Assay(s) MANDATORY ASSAYS OK
PASSES All Assays have Metabolite Assignment File (MAF) referenced OPTIONAL FILES OK
PASSES Metabolite Assignment File (MAF) is present in Study folder MANDATORY FILES OK
PASSES Metabolite Assignment File (MAF) has correct format MANDATORY FILES OK
PASSES Metabolite Identification File (MAF) content MANDATORY FILES OK
PASSES ISA-Tab investigation file check MANDATORY ISATAB OK

Pathways - Assay  1



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  2



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  3



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  4



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)