Summary of Study ST002240

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001429. The data can be accessed directly via it's Project DOI: 10.21228/M80X3B This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002240
Study TitleUse of HRMS and Dual Isotope Labels to Resolve Difficult-to Measure Fluxes
Study TypeStable isotope enriched Metabolomics
Study SummaryData analysis and mass spectrometry tools have advanced significantly in the last decade. This ongoing revolution has elevated the status of analytical chemistry within the big-data omics era. High resolution mass spectrometers (HRMS) can now distinguish different metabolites with mass to charge ratios (i.e. m/z) that differ by 0.01 Da or less. This unprecedented level of resolution not only enables identification of previously unknown compounds but also presents an opportunity to establish active metabolic pathways through quantification of isotope enrichment. Studies with stable isotope tracers continue to contribute to our knowledge of biological pathways in human, plant and bacterial species, however most current studies have been based on targeted analyses. The capacity of HRMS to resolve near-overlapping isotopologues and identify compounds with high mass precision presents a strategy to assess ‘active’ pathways de novo from data generated in an untargeted way, that is blind to the metabolic network and therefore unbiased. Currently, identifying metabolic features, enriched with stable isotopes, at an ‘omics’ level remains an experimental bottleneck, limiting our capacity to understand biological network operation at the metabolic level. We developed data analysis tools that: i) use labeling information and exact mass to determine the elemental composition of each isotopically enriched ion, ii) apply correlation-based approaches to cluster metabolite peaks with similar patterns of isotopic labels and, iii) leverage this information to build directed metabolic networks de novo. Using Camelina sativa, an emerging oilseed model, we demonstrate the power of stable isotope labeling in combination with imaging and HRMS to reconstruct lipid metabolic networks in developing seeds and are currently addressing questions about lipid and central metabolism. Tools developed in this study will have a broader application to assess context specific operation of metabolic pathways.
Institute
Donald Danforth Plant Science Center
DepartmentAllen/USDA lab
LaboratoryAllen Lab
Last NameShrikaar
First NameKambhampati
Address975 North Warson road, St. Louis, MO 63132
Emailskambhampati@danforthcenter.org
Phone3144025550
Submit Date2022-07-21
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2022-08-17
Release Version1
Kambhampati Shrikaar Kambhampati Shrikaar
https://dx.doi.org/10.21228/M80X3B
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001429
Project DOI:doi: 10.21228/M80X3B
Project Title:Using stable isotopes and mass spectrometry to elucidate the dynamics of metabolic pathways
Project Type:Stable Isotope Enriched Lipidomics
Project Summary:Data analysis and mass spectrometry tools have advanced significantly in the last decade. This ongoing revolution has elevated the status of analytical chemistry within the big-data omics era. High resolution mass spectrometers (HRMS) can now distinguish different metabolites with mass to charge ratios (i.e. m/z) that differ by 0.01 Da or less. This unprecedented level of resolution not only enables identification of previously unknown compounds but also presents an opportunity to establish active metabolic pathways through quantification of isotope enrichment. Studies with stable isotope tracers continue to contribute to our knowledge of biological pathways in human, plant and bacterial species, however most current studies have been based on targeted analyses. The capacity of HRMS to resolve near-overlapping isotopologues and identify compounds with high mass precision presents a strategy to assess ‘active’ pathways de novo from data generated in an untargeted way, that is blind to the metabolic network and therefore unbiased. Currently, identifying metabolic features, enriched with stable isotopes, at an ‘omics’ level remains an experimental bottleneck, limiting our capacity to understand biological network operation at the metabolic level. We developed data analysis tools that: i) use labeling information and exact mass to determine the elemental composition of each isotopically enriched ion, ii) apply correlation-based approaches to cluster metabolite peaks with similar patterns of isotopic labels and, iii) leverage this information to build directed metabolic networks de novo. Using Camelina sativa, an emerging oilseed model, we demonstrate the power of stable isotope labeling in combination with imaging and HRMS to reconstruct lipid metabolic networks in developing seeds and are currently addressing questions about lipid and central metabolism. Tools developed in this study will have a broader application to assess context specific operation of metabolic pathways.
Institute:Donald Danforth Plant Science Center
Department:Allen/USDA lab
Laboratory:Allen lab
Last Name:Shrikaar
First Name:Kambhampati
Address:975 North Warson road, St. Louis, MO 63132
Email:skambhampati@danforthcenter.org
Phone:3144025550
Funding Source:NIH, USDA-ARS

Subject:

Subject ID:SU002326
Subject Type:Plant
Subject Species:Arabidopsis thaliana
Taxonomy ID:3702
Age Or Age Range:10 day old seedlings
Species Group:Roots

Factors:

Subject type: Plant; Subject species: Arabidopsis thaliana (Factor headings shown in green)

mb_sample_id local_sample_id Tissue type Time (hours)
SA2140900_GAT_2-neggat1_2.1 (mutant) 0
SA2140910_GAT_1-neggat1_2.1 (mutant) 0
SA2140920_GAT_3-posgat1_2.1 (mutant) 0
SA2140930_GAT_2-posgat1_2.1 (mutant) 0
SA2140940_GAT_3-neggat1_2.1 (mutant) 0
SA2140950_GAT_4-posgat1_2.1 (mutant) 0
SA2140960_GAT_1-posgat1_2.1 (mutant) 0
SA2140970_GAT_4-neggat1_2.1 (mutant) 0
SA2140982_GAT_2-neggat1_2.1 (mutant) 2
SA2140992_GAT_2-posgat1_2.1 (mutant) 2
SA2141002_GAT_3-posgat1_2.1 (mutant) 2
SA2141012_GAT_1-neggat1_2.1 (mutant) 2
SA2141022_GAT_3-neggat1_2.1 (mutant) 2
SA2141032_GAT_4-posgat1_2.1 (mutant) 2
SA2141042_GAT_4-neggat1_2.1 (mutant) 2
SA2141052_GAT_1-posgat1_2.1 (mutant) 2
SA2141064_GAT_1-posgat1_2.1 (mutant) 4
SA2141074_GAT_2-neggat1_2.1 (mutant) 4
SA2141084_GAT_4-posgat1_2.1 (mutant) 4
SA2141094_GAT_4-neggat1_2.1 (mutant) 4
SA2141104_GAT_3-neggat1_2.1 (mutant) 4
SA2141114_GAT_3-posgat1_2.1 (mutant) 4
SA2141124_GAT_2-posgat1_2.1 (mutant) 4
SA2141134_GAT_1-neggat1_2.1 (mutant) 4
SA2141146_GAT_3-posgat1_2.1 (mutant) 6
SA2141156_GAT_3-neggat1_2.1 (mutant) 6
SA2141166_GAT_4-posgat1_2.1 (mutant) 6
SA2141176_GAT_2-neggat1_2.1 (mutant) 6
SA2141186_GAT_2-posgat1_2.1 (mutant) 6
SA2141196_GAT_1-posgat1_2.1 (mutant) 6
SA2141206_GAT_1-neggat1_2.1 (mutant) 6
SA2141216_GAT_4-neggat1_2.1 (mutant) 6
SA2141228_GAT_1-posgat1_2.1 (mutant) 8
SA2141238_GAT_3-posgat1_2.1 (mutant) 8
SA2141248_GAT_2-neggat1_2.1 (mutant) 8
SA2141258_GAT_3-neggat1_2.1 (mutant) 8
SA2141268_GAT_4-posgat1_2.1 (mutant) 8
SA2141278_GAT_1-neggat1_2.1 (mutant) 8
SA2141288_GAT_4-neggat1_2.1 (mutant) 8
SA2141298_GAT_2-posgat1_2.1 (mutant) 8
SA2140500_WT_2-posWildtype (Col 0) 0
SA2140510_WT_1-negWildtype (Col 0) 0
SA2140520_WT_3-posWildtype (Col 0) 0
SA2140530_WT_4-posWildtype (Col 0) 0
SA2140540_WT_4-negWildtype (Col 0) 0
SA2140550_WT_1-posWildtype (Col 0) 0
SA2140560_WT_3-negWildtype (Col 0) 0
SA2140570_WT_2-negWildtype (Col 0) 0
SA2140582_WT_3-posWildtype (Col 0) 2
SA2140592_WT_3-negWildtype (Col 0) 2
SA2140602_WT_4-posWildtype (Col 0) 2
SA2140612_WT_2-negWildtype (Col 0) 2
SA2140622_WT_2-posWildtype (Col 0) 2
SA2140632_WT_1-negWildtype (Col 0) 2
SA2140642_WT_1-posWildtype (Col 0) 2
SA2140652_WT_4-negWildtype (Col 0) 2
SA2140664_WT_1-negWildtype (Col 0) 4
SA2140674_WT_3-posWildtype (Col 0) 4
SA2140684_WT_2-negWildtype (Col 0) 4
SA2140694_WT_3-negWildtype (Col 0) 4
SA2140704_WT_4-posWildtype (Col 0) 4
SA2140714_WT_4-negWildtype (Col 0) 4
SA2140724_WT_2-posWildtype (Col 0) 4
SA2140734_WT_1-posWildtype (Col 0) 4
SA2140746_WT_2-negWildtype (Col 0) 6
SA2140756_WT_2-posWildtype (Col 0) 6
SA2140766_WT_1-negWildtype (Col 0) 6
SA2140776_WT_3-posWildtype (Col 0) 6
SA2140786_WT_1-posWildtype (Col 0) 6
SA2140796_WT_3-negWildtype (Col 0) 6
SA2140806_WT_4-negWildtype (Col 0) 6
SA2140816_WT_4-posWildtype (Col 0) 6
SA2140828_WT_3-negWildtype (Col 0) 8
SA2140838_WT_4-posWildtype (Col 0) 8
SA2140848_WT_1-posWildtype (Col 0) 8
SA2140858_WT_4-negWildtype (Col 0) 8
SA2140868_WT_3-posWildtype (Col 0) 8
SA2140878_WT_2-negWildtype (Col 0) 8
SA2140888_WT_1-negWildtype (Col 0) 8
SA2140898_WT_2-posWildtype (Col 0) 8
Showing results 1 to 80 of 80

Collection:

Collection ID:CO002319
Collection Summary:For the metabolomics study using dual-isotope labeling, wildtype Arabidopsis ecotype Columbia seeds were grown on vertical plates at 22°C under continuous light (ca. 70 µmol m-2 s-1), on a defined nutrient medium previously described11. The medium consisted of 10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar. Ten-day old seedlings were transferred to plates containing the same medium, except the nitrogen source was replaced with 10 mM [13C5,15N2]glutamine. Root tissue was excised after exposure to medium containing labeled glutamine for 2, 4, 6 and 8h to represent time course incorporation of carbon and nitrogen into metabolism. Untreated roots were used as unlabeled (0h) controls. Each plate yielded ~100 mg of root tissue and served as a single replicate. Four replicates per sample type were collected and flash frozen using liquid N2 for total metabolite extraction.
Sample Type:Plant
Collection Method:Flash frozen in Liquid N2
Collection Location:Donald Danforth Plant Science Center
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002338
Treatment Summary:For the metabolomics study using dual-isotope labeling, wildtype Arabidopsis ecotype Columbia seeds were grown on vertical plates at 22°C under continuous light (ca. 70 µmol m-2 s-1), on a defined nutrient medium previously described11. The medium consisted of 10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar. Ten-day old seedlings were transferred to plates containing the same medium, except the nitrogen source was replaced with 10 mM [13C5,15N2]glutamine. Root tissue was excised after exposure to medium containing labeled glutamine for 2, 4, 6 and 8h to represent time course incorporation of carbon and nitrogen into metabolism. Untreated roots were used as unlabeled (0h) controls. Each plate yielded ~100 mg of root tissue and served as a single replicate. Four replicates per sample type were collected and flash frozen using liquid N2 for total metabolite extraction.

Sample Preparation:

Sampleprep ID:SP002332
Sampleprep Summary:Frozen Arabidopsis root tissue was homogenized using a tissue lyser, and extraction was carried out using 1 mL of 4:1 methanol: water (v/v) with incubation in an ultra-sonication bath for 30 min followed by shaking for 30 min at 4°C. The mixture was then centrifuged at 21,000 x g for 10 min at 4°C; supernatant was transferred into fresh tubes and evaporated to dryness using a speedvac centrifuge at ambient temperature. Dried residue was re-suspended in 200 µL of 1:1 methanol: water (v/v), filtered using 0.2 µm PTFE micro centrifuge filters and transferred to glass vials for HILIC-HRMS runs.
Processing Storage Conditions:-80℃
Extraction Method:4:1 Methanol:Water

Combined analysis:

Analysis ID AN003657 AN003658
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Agilent 1290 Infinity II Agilent 1290 Infinity II
Column SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units Intensity Intensity

Chromatography:

Chromatography ID:CH002710
Chromatography Summary:Chromatographic separation using HILIC was achieved using an Agilent 1290 Infinity II UHPLC system equipped with a SeQuant® ZIC®-HILIC (100 x 2.1 x 3.5 µm) column (EMD Millipore, Burlington, MA). Mobile phases A and B were comprised of 5 mM ammonium acetate (pH 4.0) in water and 90% acetonitrile with 0.1 % acetic acid, respectively. A flow rate of 0.3 mL min-1 was used to elute compounds with the following gradient: 87% B for 5 minutes, decreased to 55% B over the next 8 minutes and held for 2.5 minutes before returning to 87% and equilibrating the column for 3 minutes.
Instrument Name:Agilent 1290 Infinity II
Column Name:SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:40
Flow Rate:0.3 mL min-1
Internal Standard:Equisplash
Solvent A:100% water; 5 mM ammonium acetate, pH 4
Solvent B:90% acetonitrile/10% water; 0.1% acetic acid
Chromatography Type:HILIC

MS:

MS ID:MS003408
Analysis ID:AN003657
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The heated electrospray ionization (HESI) conditions used were as follows; spray voltage, 3.9 kV (ESI+), 3.5 kV (ESI-); capillary temperature, 250 °C; probe heater temperature, 450 °C; sheath gas, 30 arbitrary units; auxiliary gas, 8 arbitrary units; and S-Lens RF level, 60%. Full MS data were collected using a Q-Exactive Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific) in both positive and negative ionization mode separately from mass ranges 75-1100 m/z and 65-900 m/z, respectively, at 140,000 resolution. The automatic gain control (AGC) was set to 3 x 106 and maximum injection time (IT) used was 524 ms.
Ion Mode:POSITIVE
  
MS ID:MS003409
Analysis ID:AN003658
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The heated electrospray ionization (HESI) conditions used were as follows; spray voltage, 3.9 kV (ESI+), 3.5 kV (ESI-); capillary temperature, 250 °C; probe heater temperature, 450 °C; sheath gas, 30 arbitrary units; auxiliary gas, 8 arbitrary units; and S-Lens RF level, 60%. Full MS data were collected using a Q-Exactive Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific) in both positive and negative ionization mode separately from mass ranges 75-1100 m/z and 65-900 m/z, respectively, at 140,000 resolution. The automatic gain control (AGC) was set to 3 x 106 and maximum injection time (IT) used was 524 ms.
Ion Mode:NEGATIVE
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