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MTBLS2: Comparative LC/MS-based profiling of silver nitrate-treated Arabidopsis thaliana leaves of wild-type and cyp79B2 cyp79B3 double knockout plants

Abstract

Indole-3-acetaldoxime (IAOx) represents an early intermediate of the biosynthesis of a variety of indolic secondary metabolites including the phytoanticipin indol-3-ylmethyl glucosinolate and the phytoalexin camalexin (3-thiazol-2′-yl-indole). Arabidopsis thaliana cyp79B2 cyp79B3 double knockout plants are completely impaired in the conversion of tryptophan to indole-3-acetaldoxime and do not accumulate IAOx-derived metabolites any longer. Consequently, comparative analysis of wild-type and cyp79B2 cyp79B3 plant lines has the potential to explore the complete range of IAOx-derived indolic secondary metabolites.

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 Authors: Christoph Böttcher, Steffen Neumann

  Release date: 22-May-2012

 Status: Public

Organism(s)

Arabidopsis thaliana

  Study Design

Plant Metabolomics

Comparative LC/MS-based profiling

Pathway Elucidation

CHMO:ultra-performance liquid chromatography-mass spectrometry

untargeted metabolites

  Experimental Factors

genotype

replicate

Protocol Description
Sample collection Arabidopsis thaliana Col-0 and cyp79B2 cyp79B3 lines were grown for about six weeks after cold stratification (3 days, 4 °C) in parallel on a soil/vermiculite mixture (3/2) in a growth cabinet with 8 h light (~150 µE/m2/s) at 22 °C and 16 h dark at 20 °C to developmental stage 3.5. Plants were sprayed with an aqueous solution of silver nitrate (5 mM) in order to stimulate accumulation of indolic secondary metabolites and incubated for 48 h in a growth cabinet under the same conditions as described above. Rosette leaves of six individual plants per genotype were harvested, immediately frozen in liquid nitrogen, pooled and stored at –80 °C until analysis. Two independent experiments (biological replicates) were performed on independent sets of plants grown at different times.
Extraction Plant material was homogenized in liquid nitrogen using a pestle and mortar and aliquots of 100 +/- 5 mg were weighed into pre-cooled 2 ml, round bottom tubes. After addition of 200 µl methanol/water, 80/20 (v/v) pre-cooled at –40 °C the samples were immediately vortexed for 15 s, sonicated for 15 min at 20 °C and centrifuged for 10 min at 19000 x g. The supernatants were transferred to new 2 ml tubes and the remaining pellets subjected to a second extraction using 200 µl methanol/water, 80/20 (v/v). The combined extracts were evaporated to dryness in a vacuum centrifuge at 30 °C, thoroughly reconstituted in 200 µl methanol/water, 30/70 (v/v) and filtered using 0.2 µm PTFE syringe filters. Four extracts (technical replicates) were prepared for each of the four leaf pools.
Chromatography Chromatographic separations were performed on an Acquity UPLC system (Waters) equipped with a HSS T3 column (100 x 1.0 mm, particle size 1.8 µm, Waters) applying the following binary gradient at a flow rate of 150 µl/min: 0-1 min, isocratic 95% A (water/formic acid, 99.9/0.1 (v/v)), 5% B (acetonitrile/formic acid, 99.9/0.1 (v/v)); 1-16 min, linear from 5 to 45% B; 16-18 min, isocratic 95% B; 18-20 min, isocratic 5% B. The injection volume was 3.0 µl (full loop injection).
Mass spectrometry Eluting compounds were detected from m/z 100-1000 using a micrOTOF-Q II hybrid quadrupole time-of-flight mass spectrometer (Bruker Daltonics) equipped with an Apollo II electrospray ion source in positive and negative ion mode using following instrument settings: nebulizer gas, nitrogen, 1.4 bar; dry gas, nitrogen, 6 l/min, 190 °C; capillary, –5000 V; end plate offset, -500 V; funnel 1 RF, 200 V; funnel 2 RF, 200 V; in-source CID energy, 0 V; hexapole RF, 100 V; quadrupole ion energy, 5 eV; collision gas, nitrogen; collision energy, 7 eV; collision RF 150/350 V (timing 50/50); transfer time, 70 µs; pre pulse storage, 5 µs; pulser frequency, 10 kHz; spectra rate, 3 Hz. Mass spectra were acquired in centroid mode. Mass calibration of individual raw data files was performed on lithium formate cluster ions obtained by automatic infusion of 20 µl 10 mM lithium hydroxide in isopropanol/water/formic acid, 49.9/49.9/0.2 (v/v/v) at a gradient time of 18 min using a diverter valve.
Data transformation Raw data files were converted to mzData format using the vendor-specific CompassXport (http://www.bdal.de/) and processed using the XCMS package (http://bioconductor.org/packages/release/bioc/html/xcms.html). XCMS settings for processing LC/MS data with findPeaks.centWave() were prefilter=(3,200); snthr=5; ppm=25; peakwidth=(5,12). For alignment group.density() function with parameters minfrac=0.75 and bw=2 was used.
Metabolite identification Differential features detected in narrow retention time windows displaying a high chromatogram correlation were grouped. In combination with the raw data, individual compound mass spectra were reconstructed and features annotated as cluster ion, quasimolecular ion, or in-source fragment ion. Annotated lists of all differential feature sets are provided in Supplemental DataSet S5 of DOI: 10.1007/s11306-012-0401-0 online. Afterwards, targeted CID-MS/MS experiments of quasimolecular ions (MS2) were performed using QTOF-MS. Based on accurate mass measurements, putative elemental compositions were calculated, filtered by isotope abundance and the number of double bond equivalents and electron parity [1], and checked for consistency in relation to elemental compositions calculated for fragment ions and observed neutral losses [2]. Mass spectral characterization of CID mass spectra can be found in Supplemental Data Set S6 (DOI: 10.1007/s11306-012-0401-0) online. Identification was based on reference spectra in MassBank (MSI Level 2 and 3), and in some cases on authentic standards (MSI Level 1).

Ref:
[1] Kind, T., & Fiehn, O. (2006). Metabolomic database annotations via query of elemental compositions: Mass accuracy is insufficient even at less than 1 ppm. BMC Bioinformatics, 7(1), 234. PMID: 16646969 [2] Konishi Y1, Kiyota T, Draghici C, Gao JM, Yeboah F, Acoca S, Jarussophon S, Purisima E. Molecular formula analysis by an MS/MS/MS technique to expedite dereplication of natural products. Anal Chem. 2007 Feb 1;79(3):1187-97. PMID: 17263353
Source Name Organism Organism part Protocol REF Sample Name genotype replicate
IPB Halle.Group-1.Subject-1 Arabidopsis thaliana rosette leaf Sample collection Ex1-Col0-48h-Ag-1 Col-0 Exp1
IPB Halle.Group-1.Subject-2 Arabidopsis thaliana rosette leaf Sample collection Ex1-Col0-48h-Ag-2 Col-0 Exp1
IPB Halle.Group-1.Subject-3 Arabidopsis thaliana rosette leaf Sample collection Ex1-Col0-48h-Ag-3 Col-0 Exp1
IPB Halle.Group-1.Subject-4 Arabidopsis thaliana rosette leaf Sample collection Ex1-Col0-48h-Ag-4 Col-0 Exp1
IPB Halle.Group-2.Subject-1 Arabidopsis thaliana rosette leaf Sample collection Ex1-cyp79-48h-Ag-1 cyp79 Exp1
IPB Halle.Group-2.Subject-2 Arabidopsis thaliana rosette leaf Sample collection Ex1-cyp79-48h-Ag-2 cyp79 Exp1
IPB Halle.Group-2.Subject-3 Arabidopsis thaliana rosette leaf Sample collection Ex1-cyp79-48h-Ag-3 cyp79 Exp1
IPB Halle.Group-2.Subject-4 Arabidopsis thaliana rosette leaf Sample collection Ex1-cyp79-48h-Ag-4 cyp79 Exp1
IPB Halle.Group-3.Subject-1 Arabidopsis thaliana rosette leaf Sample collection Ex2-Col0-48h-Ag-1 Col-0 Exp2
IPB Halle.Group-3.Subject-2 Arabidopsis thaliana rosette leaf Sample collection Ex2-Col0-48h-Ag-2 Col-0 Exp2
IPB Halle.Group-3.Subject-3 Arabidopsis thaliana rosette leaf Sample collection Ex2-Col0-48h-Ag-3 Col-0 Exp2
IPB Halle.Group-3.Subject-4 Arabidopsis thaliana rosette leaf Sample collection Ex2-Col0-48h-Ag-4 Col-0 Exp2
IPB Halle.Group-4.Subject-1 Arabidopsis thaliana rosette leaf Sample collection Ex2-cyp79-48h-Ag-1 cyp79 Exp2
IPB Halle.Group-4.Subject-2 Arabidopsis thaliana rosette leaf Sample collection Ex2-cyp79-48h-Ag-2 cyp79 Exp2
IPB Halle.Group-4.Subject-3 Arabidopsis thaliana rosette leaf Sample collection Ex2-cyp79-48h-Ag-3 cyp79 Exp2
IPB Halle.Group-4.Subject-4 Arabidopsis thaliana rosette leaf Sample collection Ex2-cyp79-48h-Ag-4 cyp79 Exp2

Assay 

Assay file name: a_mtbl2_metabolite profiling_mass spectrometry.txt
Measurement: metabolite profiling
Technology: mass spectrometry
Platform: micrOTOF-Q II ESI TOF (Bruker)

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 genotype replicate
Ex1-Col0-48h-Ag-1 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex1-Col0-48h-Ag-1 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex1-Col0-48h-Ag-1_1-A,1_01_9818 MSpos-Ex1-Col0-48h-Ag-1_1-A,1_01_9818.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv Col-0 Exp1
Ex1-Col0-48h-Ag-2 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex1-Col0-48h-Ag-2 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex1-Col0-48h-Ag-2_1-A,1_01_9820 MSpos-Ex1-Col0-48h-Ag-2_1-A,1_01_9820.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv Col-0 Exp1
Ex1-Col0-48h-Ag-3 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex1-Col0-48h-Ag-3 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex1-Col0-48h-Ag-3_1-A,1_01_9822 MSpos-Ex1-Col0-48h-Ag-3_1-A,1_01_9822.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv Col-0 Exp1
Ex1-Col0-48h-Ag-4 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex1-Col0-48h-Ag-4 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex1-Col0-48h-Ag-4_1-A,1_01_9824 MSpos-Ex1-Col0-48h-Ag-4_1-A,1_01_9824.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv Col-0 Exp1
Ex1-cyp79-48h-Ag-1 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex1-cyp79-48h-Ag-1 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex1-cyp79-48h-Ag-1_1-B,1_01_9819 MSpos-Ex1-cyp79-48h-Ag-1_1-B,1_01_9819.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv cyp79 Exp1
Ex1-cyp79-48h-Ag-2 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex1-cyp79-48h-Ag-2 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex1-cyp79-48h-Ag-2_1-B,2_01_9821 MSpos-Ex1-cyp79-48h-Ag-2_1-B,2_01_9821.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv cyp79 Exp1
Ex1-cyp79-48h-Ag-3 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex1-cyp79-48h-Ag-3 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex1-cyp79-48h-Ag-3_1-B,1_01_9823 MSpos-Ex1-cyp79-48h-Ag-3_1-B,1_01_9823.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv cyp79 Exp1
Ex1-cyp79-48h-Ag-4 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex1-cyp79-48h-Ag-4 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex1-cyp79-48h-Ag-4_1-B,2_01_9825 MSpos-Ex1-cyp79-48h-Ag-4_1-B,2_01_9825.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv cyp79 Exp1
Ex2-Col0-48h-Ag-1 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex2-Col0-48h-Ag-1 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex2-Col0-48h-Ag-1_1-A,2_01_9827 MSpos-Ex2-Col0-48h-Ag-1_1-A,2_01_9827.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv Col-0 Exp2
Ex2-Col0-48h-Ag-2 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex2-Col0-48h-Ag-2 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex2-Col0-48h-Ag-2_1-A,3_01_9829 MSpos-Ex2-Col0-48h-Ag-2_1-A,3_01_9829.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv Col-0 Exp2
Ex2-Col0-48h-Ag-3 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex2-Col0-48h-Ag-3 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex2-Col0-48h-Ag-3_1-A,4_01_9831 MSpos-Ex2-Col0-48h-Ag-3_1-A,4_01_9831.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv Col-0 Exp2
Ex2-Col0-48h-Ag-4 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex2-Col0-48h-Ag-4 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex2-Col0-48h-Ag-4_1-A,2_01_9833 MSpos-Ex2-Col0-48h-Ag-4_1-A,2_01_9833.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv Col-0 Exp2
Ex2-cyp79-48h-Ag-1 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex2-cyp79-48h-Ag-1 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex2-cyp79-48h-Ag-1_1-B,3_01_9828 MSpos-Ex2-cyp79-48h-Ag-1_1-B,3_01_9828.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv cyp79 Exp2
Ex2-cyp79-48h-Ag-2 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex2-cyp79-48h-Ag-2 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex2-cyp79-48h-Ag-2_1-B,4_01_9830 MSpos-Ex2-cyp79-48h-Ag-2_1-B,4_01_9830.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv cyp79 Exp2
Ex2-cyp79-48h-Ag-3 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex2-cyp79-48h-Ag-3 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex2-cyp79-48h-Ag-3_1-B,3_01_9832 MSpos-Ex2-cyp79-48h-Ag-3_1-B,3_01_9832.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv cyp79 Exp2
Ex2-cyp79-48h-Ag-4 Extraction 200 µl methanol/water, 30/70 (v/v) none Ex2-cyp79-48h-Ag-4 Chromatography Waters ACQUITY UPLC system ACQUITY UPLC HSS T3 (1.8 µm, 1 mm x 100 mm; Waters) reverse phase Mass spectrometry positive 100-1000 Bruker micrOTOF-Q II electrospray ionization quadrupole time-of-flight Ex2-cyp79-48h-Ag-4_1-B,4_01_9834 MSpos-Ex2-cyp79-48h-Ag-4_1-B,4_01_9834.mzData Data transformation Metabolite identification m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv cyp79 Exp2
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List of study files   Subset

File
m_mtbl2_metabolite profiling_mass spectrometry_v2_maf.tsv
MSpos-Ex1-Col0-48h-Ag-1_1-A,1_01_9818.mzData
MSpos-Ex1-Col0-48h-Ag-4_1-A,1_01_9824.mzData
MSpos-Ex1-cyp79-48h-Ag-1_1-B,1_01_9819.mzData
MSpos-Ex1-cyp79-48h-Ag-3_1-B,1_01_9823.mzData
MSpos-Ex1-Col0-48h-Ag-2_1-A,1_01_9820.mzData
MSpos-Ex1-cyp79-48h-Ag-2_1-B,2_01_9821.mzData
MSpos-Ex1-Col0-48h-Ag-3_1-A,1_01_9822.mzData
MSpos-Ex2-Col0-48h-Ag-1_1-A,2_01_9827.mzData
MSpos-Ex2-Col0-48h-Ag-3_1-A,4_01_9831.mzData
MSpos-Ex1-cyp79-48h-Ag-4_1-B,2_01_9825.mzData
MSpos-Ex2-Col0-48h-Ag-4_1-A,2_01_9833.mzData
MSpos-Ex2-Col0-48h-Ag-2_1-A,3_01_9829.mzData
MSpos-Ex2-cyp79-48h-Ag-1_1-B,3_01_9828.mzData
MSpos-Ex2-cyp79-48h-Ag-2_1-B,4_01_9830.mzData
a_mtbl2_metabolite profiling_mass spectrometry.txt
a_mtbl2_metabolite profiling_mass spectrometry_maf.csv
s_MTBL2.txt
MSpos-Ex2-cyp79-48h-Ag-4_1-B,4_01_9834.mzData
MSpos-Ex2-cyp79-48h-Ag-3_1-B,3_01_9832.mzData
metexplore_mapping.json
i_Investigation.txt
MSpos-Ex1-Col0-48h-Ag-1_1-A,1_01_9818.d
MSpos-Ex1-Col0-48h-Ag-2_1-A,1_01_9820.d
MSpos-Ex1-Col0-48h-Ag-3_1-A,1_01_9822.d
audit
MSpos-Ex1-Col0-48h-Ag-4_1-A,1_01_9824.d
MSpos-Ex1-cyp79-48h-Ag-1_1-B,1_01_9819.d
MSpos-Ex1-cyp79-48h-Ag-2_1-B,2_01_9821.d
MSpos-Ex1-cyp79-48h-Ag-3_1-B,1_01_9823.d
MSpos-Ex1-cyp79-48h-Ag-4_1-B,2_01_9825.d
MSpos-Ex2-Col0-48h-Ag-1_1-A,2_01_9827.d
MSpos-Ex2-Col0-48h-Ag-2_1-A,3_01_9829.d
MSpos-Ex2-Col0-48h-Ag-3_1-A,4_01_9831.d
MSpos-Ex2-Col0-48h-Ag-4_1-A,2_01_9833.d
MSpos-Ex2-cyp79-48h-Ag-1_1-B,3_01_9828.d
MSpos-Ex2-cyp79-48h-Ag-2_1-B,4_01_9830.d
MSpos-Ex2-cyp79-48h-Ag-3_1-B,3_01_9832.d
MSpos-Ex2-cyp79-48h-Ag-4_1-B,4_01_9834.d

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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
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PASSES Organism part MANDATORY ORGANISM OK
PASSES Study Factors MANDATORY FACTORS OK
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PASSES Assay has raw files referenced MANDATORY FILES OK
PASSES Assay referenced raw files detection in filesystem MANDATORY FILES OK
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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 



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)