Summary of Study ST002239

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 IDST002239
Study TitleInsights into plant lipid metabolism using stable isotopes and high resolution mass spectrometry
Study TypeStable isotope enriched lipidomics
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
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:SU002325
Subject Type:Plant
Subject Species:Camelina Sativa
Age Or Age Range:10 days after fertilization
Species Group:Developing seeds

Factors:

Subject type: Plant; Subject species: Camelina Sativa (Factor headings shown in green)

mb_sample_id local_sample_id Tissue type Time (hours)
SA2139780_Cotyledon_1-posCotyledon 0
SA2139790_Cotyledon_3-negCotyledon 0
SA2139800_Cotyledon_3-posCotyledon 0
SA2139810_Cotyledon_1-negCotyledon 0
SA2139820_Cotyledon_2-posCotyledon 0
SA2139830_Cotyledon_2-negCotyledon 0
SA21398416_Cotyledon_3-posCotyledon 16
SA21398516_Cotyledon_3-negCotyledon 16
SA21398616_Cotyledon_2-negCotyledon 16
SA21398716_Cotyledon_1-posCotyledon 16
SA21398816_Cotyledon_2-posCotyledon 16
SA21398916_Cotyledon_1-negCotyledon 16
SA2139902_Cotyledon_3-posCotyledon 2
SA2139912_Cotyledon_2-negCotyledon 2
SA2139922_Cotyledon_3-negCotyledon 2
SA2139932_Cotyledon_1-posCotyledon 2
SA2139942_Cotyledon_2-posCotyledon 2
SA2139952_Cotyledon_1-negCotyledon 2
SA21399632_Cotyledon_3-posCotyledon 32
SA21399732_Cotyledon_3-negCotyledon 32
SA21399832_Cotyledon_2-negCotyledon 32
SA21399932_Cotyledon_2-posCotyledon 32
SA21400032_Cotyledon_1-posCotyledon 32
SA21400132_Cotyledon_1-negCotyledon 32
SA2140024_Cotyledon_3-negCotyledon 4
SA2140034_Cotyledon_3-posCotyledon 4
SA2140044_Cotyledon_1-negCotyledon 4
SA2140054_Cotyledon_2-negCotyledon 4
SA2140064_Cotyledon_1-posCotyledon 4
SA2140074_Cotyledon_2-posCotyledon 4
SA2140088_Cotyledon_1-negCotyledon 8
SA2140098_Cotyledon_2-posCotyledon 8
SA2140108_Cotyledon_3-negCotyledon 8
SA2140118_Cotyledon_3-posCotyledon 8
SA2140128_Cotyledon_2-negCotyledon 8
SA2140138_Cotyledon_1-posCotyledon 8
SA2140140_EA_2-negEmbyo axis 0
SA2140150_EA_3-negEmbyo axis 0
SA2140160_EA_2-posEmbyo axis 0
SA2140170_EA_3-posEmbyo axis 0
SA2140180_EA_1-negEmbyo axis 0
SA2140190_EA_1-posEmbyo axis 0
SA21402016_EA_2-posEmbyo axis 16
SA21402116_EA_2-negEmbyo axis 16
SA21402216_EA_3-negEmbyo axis 16
SA21402316_EA_1-negEmbyo axis 16
SA21402416_EA_3-posEmbyo axis 16
SA21402516_EA_1-posEmbyo axis 16
SA2140262_EA_1-negEmbyo axis 2
SA2140272_EA_3-negEmbyo axis 2
SA2140282_EA_2-posEmbyo axis 2
SA2140292_EA_1-posEmbyo axis 2
SA2140302_EA_3-posEmbyo axis 2
SA2140312_EA_2-negEmbyo axis 2
SA21403232_EA_1-posEmbyo axis 32
SA21403332_EA_2-posEmbyo axis 32
SA21403432_EA_1-negEmbyo axis 32
SA21403532_EA_2-negEmbyo axis 32
SA21403632_EA_3-posEmbyo axis 32
SA21403732_EA_3-negEmbyo axis 32
SA2140384_EA_2-negEmbyo axis 4
SA2140394_EA_3-negEmbyo axis 4
SA2140404_EA_2-posEmbyo axis 4
SA2140414_EA_1-posEmbyo axis 4
SA2140424_EA_3-posEmbyo axis 4
SA2140434_EA_1-negEmbyo axis 4
SA2140448_EA_1-posEmbyo axis 8
SA2140458_EA_3-negEmbyo axis 8
SA2140468_EA_3-posEmbyo axis 8
SA2140478_EA_2-negEmbyo axis 8
SA2140488_EA_2-posEmbyo axis 8
SA2140498_EA_1-negEmbyo axis 8
Showing results 1 to 72 of 72

Collection:

Collection ID:CO002318
Collection Summary:Plant growth and culture conditions: Plants were grown in greenhouses with day/night temperature maintained at 22/20°C, 40-50% relative humidity, and 16h day/8h night photoperiod. Intact siliques during the seed filling growth stage (15 days after fertilization) were excised and placed in sterile media containing a modified Linsmaier and Skoog medium23,24 with Gamborg’s vitamins (Sigma) and 5 mM MES buffer adjusted to pH 5.8. Fifty mM [U-13C6]glucose was used as labeled substrate, and the composition of the remaining carbon and nitrogen sources represented maternal phloem composition to minimize metabolic perturbation and to maintain pseudo in vivo conditions as previously described25. Silique culturing was performed in a 96-well plate with 0.3 mL of medium and a single silique per well, under continuous light (250 µmol m-2 s-1). Tissue was collected and flash frozen immediately after each time point (2, 4, 8, 16 and 32h). Uncultured siliques excised from the maternal plant were used as unlabeled (0h) controls. Frozen tissue was sectioned, on top of dry ice, to excise embryo from the siliques and to separate cotyledons from the embryo axis. Cotyledon samples were extracted and analyzed for lipids in triplicates.
Collection Protocol Filename:13CLipids_CamelinaSeeds_Methods.docx
Sample Type:Seeds
Collection Location:Donald Danforth Plant Science Center
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002337
Treatment Summary:Plants were grown in greenhouses with day/night temperature maintained at 22/20°C, 40-50% relative humidity, and 16h day/8h night photoperiod. Intact siliques during the seed filling growth stage (15 days after fertilization) were excised and placed in sterile media containing a modified Linsmaier and Skoog medium23,24 with Gamborg’s vitamins (Sigma) and 5 mM MES buffer adjusted to pH 5.8. Fifty mM [U-13C6]glucose was used as labeled substrate, and the composition of the remaining carbon and nitrogen sources represented maternal phloem composition to minimize metabolic perturbation and to maintain pseudo in vivo conditions as previously described25. Silique culturing was performed in a 96-well plate with 0.3 mL of medium and a single silique per well, under continuous light (250 µmol m-2 s-1). Tissue was collected and flash frozen immediately after each time point (2, 4, 8, 16 and 32h). Uncultured siliques excised from the maternal plant were used as unlabeled (0h) controls. Frozen tissue was sectioned, on top of dry ice, to excise embryo from the siliques and to separate cotyledons from the embryo axis. Cotyledon samples were extracted and analyzed for lipids in triplicates.

Sample Preparation:

Sampleprep ID:SP002331
Sampleprep Summary:Frozen cotyledon samples from Camelina were homogenized using a tissue lyser and the extraction of lipids was carried out using a phase separation method previously described26. Briefly, 1 mL 7:3 methanol:chloroform (-20°C) containing the ultimateSPLASHTM ONE lipid mix (Avanti Polar lipids, Alabaster, AL) as internal standard (1:20 dilution) was added to the samples, vortexed vigorously and incubated on a rotary shaker for 2 hours at 4°C. After incubation, 500 µL of ice-cold water was added to the samples, vortexed and centrifuged at 14,000 rpm at 4°C for 10 min to achieve phase separation. The upper aqueous phase was carefully removed, 200 µL of methanol was added to the remaining organic phase containing lipids and centrifuged at 14,000 rpm for 5 min to pellet the debris. The organic phase (supernatant) was transferred to a glass tube and dried using a speedvac centrifuge. Samples were re-suspended in 200 µL of 49:49:2 acetonitrile: methanol: chloroform, filtered using 0.2 µm PTFE microcentrifuge filters and transferred to a glass vial for RPLC-HRMS analysis.
Processing Storage Conditions:-80℃
Extraction Method:methanol:chloroform:water

Combined analysis:

Analysis ID AN003655 AN003656
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Dionex UltiMate 3000 RSLCnano Dionex UltiMate 3000 RSLCnano
Column Custom C8 - Higgins Analytical Custom C8 - Higgins Analytical
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Fusion Tribrid Orbitrap Thermo Fusion Tribrid Orbitrap
Ion Mode POSITIVE NEGATIVE
Units Intensity Intensity

Chromatography:

Chromatography ID:CH002709
Chromatography Summary:Separations for lipidomics were achieved using the loading pump of a Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) operating at a flow rate of 40 µL min-1 equipped with a custom-made C8 column (100 x 0.5 x 5 µm) from Higgins Analytical Inc. (Mountain view, CA) re-packed from a nucleodur C8 Gravity column (Macherey-Nagel, Allentown, PA). Mobile phases comprised of 1% 1 M ammonium acetate, 0.1 % acetic acid in water (A) and 1% 1 M ammonium acetate, 0.1% acetic acid in 7:3 (v/v) acetonitrile: isopropanol (B). The following gradient modified from a previously described method27 to adapt to micro flow was used; 0-1 min at 55% B, 4 min at 75% B, 12 min at 89% B, 15 min at 99% B, 18 min at 99% B and 20 min at 55% B followed by equilibration up to 30 min.
Instrument Name:Dionex UltiMate 3000 RSLCnano
Column Name:Custom C8 - Higgins Analytical
Column Temperature:40
Flow Rate:0.04 mL min-1
Internal Standard:Equisplash
Solvent A:100% water; 0.1% acetic acid; 10 mM ammonium acetate
Solvent B:70% acetonitrile/30% isopropanol; 0.1% acetic acid; 10 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS003406
Analysis ID:AN003655
Instrument Name:Thermo Fusion Tribrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The eluent was sprayed on to the HESI source of an Orbitrap Fusion Lumos Tribrid MS, operated with sheath gas, 25 arbitrary units; auxiliary gas, 5 arbitrary units; ion transfer tube temperature, 300oC; vaporizer temperature, 100oC; and S-lens RF level, 60. The spray voltage was 4 kV in both positive and negative modes. Full MS data were collected for mass ranges 450-1200 m/z at 240,000 resolution from both positive and negative modes simultaneously, using polarity switch. The AGC target was set to “Standard” and the maximum IT was set to 100 ms.
Ion Mode:POSITIVE
  
MS ID:MS003407
Analysis ID:AN003656
Instrument Name:Thermo Fusion Tribrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The eluent was sprayed on to the HESI source of an Orbitrap Fusion Lumos Tribrid MS, operated with sheath gas, 25 arbitrary units; auxiliary gas, 5 arbitrary units; ion transfer tube temperature, 300oC; vaporizer temperature, 100oC; and S-lens RF level, 60. The spray voltage was 4 kV in both positive and negative modes. Full MS data were collected for mass ranges 450-1200 m/z at 240,000 resolution from both positive and negative modes simultaneously, using polarity switch. The AGC target was set to “Standard” and the maximum IT was set to 100 ms.
Ion Mode:NEGATIVE
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