MTBLS228: Untargeted extraction of metabolites 13C labeling profiles from time course labeling switch experiment

Abstract

Dynamic isotope labeling data provide crucial information about the operation of metabolic pathways, and are commonly generated via liquid chromatography mass spectrometry (LC-MS). Metabolome-wide analysis is challenging as it requires grouping of metabolite features over different samples. We developed DynaMet for fully automated investigations of isotope labeling experiments from LC-high resolution MS raw data. DynaMet enables untargeted extraction of metabolite labeling profiles and provides integrated tools for expressive data visualization. For this study we generated labeling data of the model strain Bacillus methanolicus from 13C methanol resulting in complex spectra in multi carbon compounds. Analysis of two biological replicates revealed high robustness and reproducibility of the pipeline. In total, DynaMet extracted 386 features showing dynamic labeling within ten minutes. Of these features, 357 could be fitted by implemented kinetic models. Feature identification against KEGG database resulted in 247 matches covering multiple pathways of core metabolism and major biosynthetic routes. The study results reported here are only a summary. To reproduce the complete results including plots, DynaMet software is required.

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 Authors: Patrick Kiefer

  Release date: 19-Oct-2015

 Status: Public

Organism(s)

Bacillus methanolicus

  Study Design

FIX:isotope method

nanoscale high performance liquid chromatography mass spectrometry

untargeted metabolites

DynaMet

  Experimental Factors

incubation time

Protocol Description
Sample collection B. Methanolicus MGA3 cells were sampled at OD600 between 1 and 1.5. To perform 13C labeling , 0.5 ml of B. methanolicus MGA3 culture grown in bioreactors with naturally labeled methanol was transferred into 50 ml Falcon tubes containing 4.5 ml of temperature adjusted (50 °C) MSV medium with 0.5 M 99% 13C methanol. Samples in tubes were continuously mixed and incubated for 5 s, 10 s, 20 s, 30 s, 60 s, 120 s, 300 s, and 600 s at the respective temperatures before the entire 5 ml was transferred onto a 0.2 µm polyethylensulfon (PESU) filter (Sartorius Stedium). Cells were vacuum filtered and immediately washed with 10 ml warm MilliQ water (20 °C) to remove culture medium.
Extraction For quenching and metabolite extraction, filters with cells were transferred into 8 ml quenching solution (mixture of acetonitrile, methanol and 0.5 M formic acid at a 60:20:20 ratio) at -20 °C, incubated for 10 minutes on ice and sonified 3 times for 10 s based on a protocol adapted from [1]. Samples were snap frozen in liquid N2, lyophilized and stored at -20 °C until analysis. Prior to analysis, samples were resuspended with MilliQ water to a final biomass concentration of 1 µg/µl. Biomass content of the samples was calculated based on sample volume and corresponding culture OD600.

Ref:
[1] Kiefer P, Schmitt U, Vorholt JA. eMZed: an open source framework in Python for rapid and interactive development of LC/MS data analysis workflows. Bioinformatics. 2013 Apr 1;29(7):963-4. doi: 10.1093/bioinformatics/btt080. PMID: 23418185
Chromatography Analysis of the prepared 13C-labeled experiment samples was performed using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) with a split-free nanoLC Ultra system (Eksigent, Dublin, CA) hyphenated to an LTQ-Orbitrap mass spectrometer (ThermoFisher Scientific, San Jose, CA) by nanoelectrospray ionization as described previously with slight modifications [1]. Separation of the metabolites was performed on a C18 column (Dr. Maisch HPLC Markensäule, Reprosil-Gold 120, 100 mm x 0.1 mm, Morvay Analytik GmbH) as the stationary phase with a tributylamine (TBA) solution as the ion-pairing reagent and methanol (solvent B) as the eluent. The TBA solution (solvent A) was obtained by dissolving 1.7 mM TBA in 1.5 mM acetic acid; the pH was adjusted to 9.0 with 6 M ammonium hydroxide. Solvent B (methanol) varied according to the gradient under a flow rate of 400 nl/min as follows: 0 min, 3%; 30 min, 90%; 35 min, 90%; 36 min, 3%; and 45 min, 3%. The injection volume was 1 µl.

Ref:
[1] Kiefer, P., Delmotte, N., and Vorholt, J. A. (2011) Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis. Anal Chem 83, 850-855. doi: 10.1021/ac102445r. PMID:21166460
Mass spectrometry LC–HRMS analysis was performed applying a nanoscale ion-pair reversed-phase HPLC–MS method [1] with an LTQ-Orbitrap instrument operating in negative FT mode at unit resolution of 60,000 (at m/z 400) and with a scan range of m/z 150-850. The Orbitrap Parameters can be found in a text file in the study folder.

Ref:
[1] Kiefer P, Delmotte N, Vorholt JA. Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis. Anal Chem. 2011 Feb 1;83(3):850-5. doi: 10.1021/ac102445r. PMID:21166460.
Data transformation Conversion from Thermo raw data files into mzML using Proteowizard tool msconvert (http://proteowizard.sourceforge.net/tools.shtml). All further Processing was performed with DynaMet app, an extension package from emzed (installation can be found at http://emzed.ethz.ch/installation.html). After installation of emzed, DynaMet can be directly installed by typing following command in the IPython console: !pip install dynamet. An overview of all processing steps is shown in the file 'Data Transformation Protocol.txt' which is available in the study folder.
Metabolite identification Metabolite identification was performed using identification module of Dynamet software. To match features to data base, monoisotopic masses of features are calculated from possible adducts mass shifts. In case of successful adduct grouping the assigned monoisotopic mass is used to search for masses in database within user defined maximal deviation whereby matches are further restricted by estimation of metabolites possible number of carbon atoms. We estimated the number of carbon atoms from mass isotopologue pattern of natural labeled, and if provided, from a mixture of natural labeled and uniformly 13C labeled cell extract. As a general restriction the range of the possible number of carbon atoms is calculated for each metabolite, whereby the lower limit is defined by the number of 13C related mass isotopologues grouped per feature and the upper limit by the compound with similar mass present in the Pubchem database containing most number of carbon atoms. Depending on the consistency and reliability of the results obtained by applied estimation methods, each estimation gets a quality score (q-score) ranging from 0 to 15, and best scored estimation is selected. For the current study the KEGG database was used.

For each compound in each sample we determined the summed number of labeled Carbon atoms:
n_13C=(f_M0 * f_M1*1 + f_Mn *n)
where n is number of C atoms, f_Mi is f_Mi=A_Mi/A_tot and A_Mi is ith isotopologue of metabolite i, A_tot is summed area of all isotopologues, A_tot=summed area of all isotopologue of a feauture where m is the metabolites number of carbon atoms.
Source Name Organism Variant Organism part Protocol REF Sample Name incubation time Unit
20131126_024_JM_BM_exp12-10-18_S1_0s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_024_JM_BM_exp12-10-18_S1_0s 0 second
20131126_026_JM_BM_exp12-10-18_S2_5s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_026_JM_BM_exp12-10-18_S2_5s 5 second
20131126_028_JM_BM_exp12-10-18_S3_10s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_028_JM_BM_exp12-10-18_S3_10s 10 second
20131126_030_JM_BM_exp12-10-18_S4_20s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_030_JM_BM_exp12-10-18_S4_20s 20 second
20131126_032_JM_BM_exp12-10-18_S5_30s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_032_JM_BM_exp12-10-18_S5_30s 30 second
20131126_034_JM_BM_exp12-10-18_S6_60s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_034_JM_BM_exp12-10-18_S6_60s 60 second
20131126_036_JM_BM_exp12-10-18_S7_120s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_036_JM_BM_exp12-10-18_S7_120s 120 second
20131126_038_JM_BM_exp12-10-18_S8_300s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_038_JM_BM_exp12-10-18_S8_300s 300 second
20131126_040_JM_BM_exp12-10-18_S9_600s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131126_040_JM_BM_exp12-10-18_S9_600s 600 second
20131204_072_JM_BM_internal_standard Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131204_072_JM_BM_internal_standard second
20131209_103_JM_BM_exp12-09-26_S1_0s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_103_JM_BM_exp12-09-26_S1_0s 0 second
20131209_105_JM_BM_exp12-09-26_S2_5s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_105_JM_BM_exp12-09-26_S2_5s 5 second
20131209_107_JM_BM_exp12-09-26_S3_10s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_107_JM_BM_exp12-09-26_S3_10s 10 second
20131209_109_JM_BM_exp12-09-26_S4_20s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_109_JM_BM_exp12-09-26_S4_20s 20 second
20131209_111_JM_BM_exp12-09-26_S5_30s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_111_JM_BM_exp12-09-26_S5_30s 30 second
20131209_113_JM_BM_exp12-09-26_S6_60s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_113_JM_BM_exp12-09-26_S6_60s 60 second
20131209_115_JM_BM_exp12-09-26_S7_120s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_115_JM_BM_exp12-09-26_S7_120s 120 second
20131209_117_JM_BM_exp12-09-26_S8_300s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_117_JM_BM_exp12-09-26_S8_300s 300 second
20131209_119_JM_BM_exp12-09-26_S9_600s Bacillus methanolicus Bacillus methanolicus MGA3 whole organism Sample collection 20131209_119_JM_BM_exp12-09-26_S9_600s 600 second

Assay 

Assay file name: a_dli_metabolite_profiling_mass_spectrometry.txt
Measurement: metabolite profiling
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
20131126_024_JM_BM_exp12-10-18_S1_0s Extraction sample diluted 1:20 with solvent A none 20131126_024_JM_BM_exp12-10-18_S1_0s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_024_JM_BM_exp12-10-18_S1_0s 20131126_024_JM_BM_exp12-10-18_S1_0s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131126_026_JM_BM_exp12-10-18_S2_5s Extraction sample diluted 1:20 with solvent A none 20131126_026_JM_BM_exp12-10-18_S2_5s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_026_JM_BM_exp12-10-18_S2_5s 20131126_026_JM_BM_exp12-10-18_S2_5s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131126_028_JM_BM_exp12-10-18_S3_10s Extraction sample diluted 1:20 with solvent A none 20131126_028_JM_BM_exp12-10-18_S3_10s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_028_JM_BM_exp12-10-18_S3_10s 20131126_028_JM_BM_exp12-10-18_S3_10s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131126_030_JM_BM_exp12-10-18_S4_20s Extraction sample diluted 1:20 with solvent A none 20131126_030_JM_BM_exp12-10-18_S4_20s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_030_JM_BM_exp12-10-18_S4_20s 20131126_030_JM_BM_exp12-10-18_S4_20s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131126_032_JM_BM_exp12-10-18_S5_30s Extraction sample diluted 1:20 with solvent A none 20131126_032_JM_BM_exp12-10-18_S5_30s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_032_JM_BM_exp12-10-18_S5_30s 20131126_032_JM_BM_exp12-10-18_S5_30s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131126_034_JM_BM_exp12-10-18_S6_60s Extraction sample diluted 1:20 with solvent A none 20131126_034_JM_BM_exp12-10-18_S6_60s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_034_JM_BM_exp12-10-18_S6_60s 20131126_034_JM_BM_exp12-10-18_S6_60s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131126_036_JM_BM_exp12-10-18_S7_120s Extraction sample diluted 1:20 with solvent A none 20131126_036_JM_BM_exp12-10-18_S7_120s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_036_JM_BM_exp12-10-18_S7_120s 20131126_036_JM_BM_exp12-10-18_S7_120s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131126_038_JM_BM_exp12-10-18_S8_300s Extraction sample diluted 1:20 with solvent A none 20131126_038_JM_BM_exp12-10-18_S8_300s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_038_JM_BM_exp12-10-18_S8_300s 20131126_038_JM_BM_exp12-10-18_S8_300s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131126_040_JM_BM_exp12-10-18_S9_600s Extraction sample diluted 1:20 with solvent A none 20131126_040_JM_BM_exp12-10-18_S9_600s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131126_040_JM_BM_exp12-10-18_S9_600s 20131126_040_JM_BM_exp12-10-18_S9_600s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131204_072_JM_BM_internal_standard Extraction sample diluted 1:20 with solvent A none 20131204_072_JM_BM_internal_standard Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131204_072_JM_BM_internal_standard 20131204_072_JM_BM_internal_standard.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_103_JM_BM_exp12-09-26_S1_0s Extraction sample diluted 1:20 with solvent A none 20131209_103_JM_BM_exp12-09-26_S1_0s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_103_JM_BM_exp12-09-26_S1_0s 20131209_103_JM_BM_exp12-09-26_S1_0s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_105_JM_BM_exp12-09-26_S2_5s Extraction sample diluted 1:20 with solvent A none 20131209_105_JM_BM_exp12-09-26_S2_5s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_105_JM_BM_exp12-09-26_S2_5s 20131209_105_JM_BM_exp12-09-26_S2_5s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_107_JM_BM_exp12-09-26_S3_10s Extraction sample diluted 1:20 with solvent A none 20131209_107_JM_BM_exp12-09-26_S3_10s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_107_JM_BM_exp12-09-26_S3_10s 20131209_107_JM_BM_exp12-09-26_S3_10s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_109_JM_BM_exp12-09-26_S4_20s Extraction sample diluted 1:20 with solvent A none 20131209_109_JM_BM_exp12-09-26_S4_20s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_109_JM_BM_exp12-09-26_S4_20s 20131209_109_JM_BM_exp12-09-26_S4_20s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_111_JM_BM_exp12-09-26_S5_30s Extraction sample diluted 1:20 with solvent A none 20131209_111_JM_BM_exp12-09-26_S5_30s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_111_JM_BM_exp12-09-26_S5_30s 20131209_111_JM_BM_exp12-09-26_S5_30s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_113_JM_BM_exp12-09-26_S6_60s Extraction sample diluted 1:20 with solvent A none 20131209_113_JM_BM_exp12-09-26_S6_60s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_113_JM_BM_exp12-09-26_S6_60s 20131209_113_JM_BM_exp12-09-26_S6_60s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_115_JM_BM_exp12-09-26_S7_120s Extraction sample diluted 1:20 with solvent A none 20131209_115_JM_BM_exp12-09-26_S7_120s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_115_JM_BM_exp12-09-26_S7_120s 20131209_115_JM_BM_exp12-09-26_S7_120s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_117_JM_BM_exp12-09-26_S8_300s Extraction sample diluted 1:20 with solvent A none 20131209_117_JM_BM_exp12-09-26_S8_300s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_117_JM_BM_exp12-09-26_S8_300s 20131209_117_JM_BM_exp12-09-26_S8_300s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
20131209_119_JM_BM_exp12-09-26_S9_600s Extraction sample diluted 1:20 with solvent A none 20131209_119_JM_BM_exp12-09-26_S9_600s Chromatography Eksigent NanoLC-Ultra 2D system Reprosil-Gold 120 C18 (3 µm, 0.1 mm x 100 mm; Dr. Maisch) reverse phase Carbon 13 Mass spectrometry negative 150-850 Thermo Scientific LTQ Orbitrap XL electrospray ionization MS:orbitrap 20131209_119_JM_BM_exp12-09-26_S9_600s 20131209_119_JM_BM_exp12-09-26_S9_600s.mzML Data transformation Metabolite identification m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
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List of study files   Subset

File
20131126_026_JM_BM_exp12-10-18_S2_5s.mzML
20131126_024_JM_BM_exp12-10-18_S1_0s.mzML
20131126_032_JM_BM_exp12-10-18_S5_30s.mzML
20131126_034_JM_BM_exp12-10-18_S6_60s.mzML
20131126_030_JM_BM_exp12-10-18_S4_20s.mzML
20131126_040_JM_BM_exp12-10-18_S9_600s.mzML
20131126_028_JM_BM_exp12-10-18_S3_10s.mzML
metexplore_mapping.json
20131204_072_JM_BM_internal_standard.mzML
20131209_103_JM_BM_exp12-09-26_S1_0s.mzML
20131126_036_JM_BM_exp12-10-18_S7_120s.mzML
20131126_038_JM_BM_exp12-10-18_S8_300s.mzML
audit
20131209_105_JM_BM_exp12-09-26_S2_5s.mzML
20131209_107_JM_BM_exp12-09-26_S3_10s.mzML
20131209_109_JM_BM_exp12-09-26_S4_20s.mzML
20131209_111_JM_BM_exp12-09-26_S5_30s.mzML
20131209_113_JM_BM_exp12-09-26_S6_60s.mzML
20131209_115_JM_BM_exp12-09-26_S7_120s.mzML
20131209_117_JM_BM_exp12-09-26_S8_300s.mzML
20131209_119_JM_BM_exp12-09-26_S9_600s.mzML
Orbitrap Parameters.txt
Data Transformation Protocol.txt
a_dli_metabolite_profiling_mass_spectrometry.txt
i_Investigation.txt
m_dli_metabolite_profiling_mass_spectrometry_v2_maf.tsv
s_DLI.txt
validation_files.json
validation_report.json

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Validations marked with (*) have been allowed by the MetaboLights Curators.
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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 



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)