Summary of Study ST000621

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 PR000454. The data can be accessed directly via it's Project DOI: 10.21228/M8CC9H 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 IDST000621
Study TitleUntargeted metabolomic changes in Chlamydomonas reinhardtii treated with lipid inducing small molecules
Study SummaryA study to investigate the effect of small molecule lipid inducing compounds that leads to hyper accumulation of lipids in N replete cells of Chlamydomonas reinhardtii. These compounds were identified through a high throughput screening designed for that purpose. During that screening, we screened 43,783 compounds and identified 367 primary hits. These 367 hits were further retested using a 8-point dilution series (from 0.25 to 30 uM) and verified the activity of 250 compounds that induce the hyper lipid accumulating phenotype in algae. Once the hit compounds were identified and confirmed, we then performed extensive chemoinformatics analysis to look for common scaffolds and identified several common substructures. We then selected 15 top performing compounds from 5 diverse structural groups and tested biochemical parameters such as growth, lipid accumulating capacity, effect on photosynthetic rates, respiration rates, oxygen consumption rates, analysis of different lipid species to quantify and identify fatty acid species using GC-MS. To understand the global changes in the metabolome, 2 structurally different compounds were selected and compared with cells grown without compounds as control for untargeted metabolomics analysis.
Institute
University of Nebraska-Lincoln
DepartmentDepartment of Biochemistry
Last NameWase
First NameNishikant
AddressDepartment of Biochemistry, 1901 VINE STREET
Emailnishikant.wase@gmail.com
Phone4023109931
Submit Date2017-06-16
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2019-01-22
Release Version1
Nishikant Wase Nishikant Wase
https://dx.doi.org/10.21228/M8CC9H
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000454
Project DOI:doi: 10.21228/M8CC9H
Project Title:Untargeted metabolomic changes in Chlamydomonas reinhardtii treated with lipid inducing small molecules
Project Summary:A study to investigate the effect of small molecule lipid inducing compounds that leads to hyper accumulation of lipids in N replete cells of Chlamydomonas reinhardtii. These compounds were identified through a high throughput screening designed for that purpose. During that screening, we screened 43,783 compounds and identified 367 primary hits. These 367 hits were further retested using a 8-point dilution series (from 0.25 to 30 uM) and verified the activity of 250 compounds that induce the hyper lipid accumulating phenotype in algae. Once the hit compounds were identified and confirmed, we then performed extensive chemoinformatics analysis to look for common scaffolds and identified several common substructures. We then selected 15 top performing compounds from 5 diverse structural groups and tested biochemical parameters such as growth, lipid accumulating capacity, effect on photosynthetic rates, respiration rates, oxygen consumption rates, analysis of different lipid species to quantify and identify fatty acid species using GC-MS. To understand the global changes in the metabolome, 2 structurally different compounds were selected and compared with cells grown without compounds as control for untargeted metabolomics analysis.
Institute:University of Nebraska-Lincoln
Department:Biochemistry
Laboratory:FATTTLab
Last Name:Wase
First Name:Nishikant
Address:1901 Beadle Center, Vine Street, 1901 VINE STREET, Lincoln, NE, 68588-0664, USA
Email:nishikant.wase@gmail.com
Phone:4023109931
Funding Source:NCESR-704, Nebraska Center for Energy Science Research; EPS-1004094 and 1264409, National Science Foundation ; NSF CBET : 1402896, National Science Foundation
Contributors:Nishikant Wase, Jiri Adamec, Ron Cerny, Girish Rasineni, Paul N Black, Concetta DiRusso

Subject:

Subject ID:SU000644
Subject Type:Photosynthetic organism
Subject Species:Chlamydomonas reinhardtii
Taxonomy ID:3055
Genotype Strain:CC125 Wild type
Species Group:Microorganism

Factors:

Subject type: Photosynthetic organism; Subject species: Chlamydomonas reinhardtii (Factor headings shown in green)

mb_sample_id local_sample_id Group
SA035012CD06241608cmp_030
SA035013CD06241609cmp_030
SA035014CD06241607cmp_030
SA035015CD06241620cmp_784
SA035016CD06241619cmp_784
SA035017CD06241621cmp_784
SA035009CD06241603Control
SA035010CD06241601BControl
SA035011CD06241602Control
Showing results 1 to 9 of 9

Collection:

Collection ID:CO000638
Collection Summary:Triplicate cultures were grown and treated as indicated and harvested at 72 h. The harvested cells were freeze-dried and weighted 10 ± 0.5 mg dry powder. To extract the metabolites, 500 uL of 75% MeOH in 0.15 M NaCl was added and metabolites were extracted using bead beating 50 Hz for 5 min. The homogenate was sonicated in water bath for 5 min and then 300 uL of 0.15 M NaCl was added and then 1 mL of chloroform:methanol (1:2) with 0.01% BHT was added. Samples were homogenized and allowed to phase separate on ice for 30 mins. Finally tubes were centrifuged for 10 min to separate the polar and non-polar layer. Top polar layer was removed and an aliquot of 200 uL was used for analysis. Samples were evaporated to dryness in SpeedVac and metabolites were reconstituted in 0.03% formic acid in analytical-grade water. Debris was removed via centrifugation and the supernatant was subjected to UPLC-MS analysis.
Sample Type:Photosynthetic organism
Collection Method:Centrifugation from suspension culture.
Collection Location:FATTTLab Department of Biochemistry, Univ of Nebraska-Lincoln
Storage Conditions:After harvest, cells were kept at -80 C until extraction.
Collection Vials:2 mL Eppendorf tubes

Treatment:

Treatment ID:TR000658
Treatment Summary:For compound treatment, cells from mid-log phase culture were harvested, washed once with fresh sterile TAP media and inoculated at starting density of 5 x 105 cells/mL. Thus for the current experiment 3 different treatment conditions were generated. Cells without compound treatment were negative control for the lipid accumulation and 2 compounds were used to generate treatment conditions. All compounds were used at a final concentration of 5 M (this concentration was determined using a dose-response curve). For all cultures, 250 mL Erlenmeyer flasks with rubber stopper adopted to facilitate gas exchange were used and maintained in horizontal orbital shaking growing chamber (Innova 43; New Brunswick). At the end of 72h, cells were harvested, media was removed via centrifugation and biomass was stored at -80 C until metabolite extraction.
Cell Media:Tris-Acetate Phoshphate (TAP) media.

Sample Preparation:

Sampleprep ID:SP000651
Sampleprep Summary:Triplicate cultures were grown and treated as indicated and harvested at 72 h. The harvested cells were freeze-dried and weighted 10 ± 0.5 mg dry powder. To extract the metabolites, 500 uL of 75% MeOH in 0.15 M NaCl was added and metabolites were extracted using bead beating 50 Hz for 5 min. The homogenate was sonicated in water bath for 5 min and then 300 uL of 0.15 M NaCl was added and then 1 mL of chloroform:methanol (1:2) with 0.01% BHT was added. Samples were homogenized and allowed to phase separate on ice for 30 mins. Finally tubes were centrifuged for 10 min to separate the polar and non-polar layer. Top polar layer was removed and an aliquot of 200 uL was used for analysis. Samples were evaporated to dryness in SpeedVac and metabolites were reconstituted in 0.03% formic acid in analytical-grade water. Debris was removed via centrifugation and the supernatant was subjected to UPLC-MS analysis
Extraction Method:Bead beating

Combined analysis:

Analysis ID AN000953
Analysis type MS
Chromatography type Reversed phase
Chromatography system Agilent 1200
Column ACE 5 C18-300 (100 x 2.1mm)
MS Type ESI
MS instrument type QTOF
MS instrument name Waters Synapt G2
Ion Mode POSITIVE
Units intensity

Chromatography:

Chromatography ID:CH000678
Instrument Name:Agilent 1200
Column Name:ACE 5 C18-300 (100 x 2.1mm)
Flow Rate:0.1 mL/min
Sample Injection:8 uL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% methanol; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS000848
Analysis ID:AN000953
Instrument Name:Waters Synapt G2
Instrument Type:QTOF
MS Type:ESI
Ion Mode:POSITIVE
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