Summary of study ST001062

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 PR000712. The data can be accessed directly via it's Project DOI: 10.21228/M8N965 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001062
Study TitleArabidopsis Nit1 knockout metabolomics
Study SummaryGlutathione (GSH) is a tripeptide that is implicated in various crucial physiological processes including redox buffering and protection against heavy metal toxicity. GSH is abundant in plants, with reported intracellular concentrations typically in the 1-10 millimolar range. Various aminotransferases can inadvertently transaminate the amino group of the γ-glutamyl moiety of GSH to produce deaminated glutathione (dGSH), a metabolite damage product. It was recently reported that an amidase known as Nit1 participates in dGSH breakdown in mammals and yeast. Plants have a hitherto uncharacterized homolog of the Nit1 amidase. We show that recombinant Arabidopsis Nit1 (At4g08790) has efficient amidase activity towards dGSH. Ablating the Arabidopsis Nit1 gene causes a massive accumulation of dGSH and other marked changes to the metabolome. All plant Nit1 sequences examined had predicted plastidial targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays show that both potential translation start codons were used and subcellular localization of GFP fusions confirmed both cytosolic and plastidial localization. Further, we show that Arabidopsis enzymes convert GSH to dGSH at a rate of 2.8 pmol min-1 mg-1 in vitro. Our data demonstrate that plants have a dGSH repair system that is directed to at least two subcellular compartments via the use of alternative translation start sites.
Institute
University of California, Davis
Last NameFolz
First NameJacob
Address451 Health Sciences Dr., Davis, CA, 95616
Emailjfolz@ucdavis.edu
Phone7155636311
Submit Date2018-09-24
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2019-03-06
Release Version1
Jacob Folz Jacob Folz
https://dx.doi.org/10.21228/M8N965
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000712
Project DOI:doi: 10.21228/M8N965
Project Title:Nit1 Arabidopsis knockout
Project Type:Untargeted Mass Spectrometry
Project Summary:Arabidopsis tissue with knockout of Nit1 gene analyzed for disruptions in metabolism. Polar and non-polar metabolites were measured using HILIC chromatography and C18 chromatography
Institute:University of California, Davis
Laboratory:Oliver Fiehn
Last Name:Folz
First Name:Jacob
Address:451 Health Science Drive, Davis, CA 95616
Email:jfolz@ucdavis.edu
Phone:7155636311
Funding Source:MCB-1153491
Contributors:Thomas D. Niehaus, Jenelle Patterson, Danny C. Alexander, Jacob S. Folz, Michal Pyc, Brian S. MacTavish, Robert T. Mullen, Oliver Fiehn, Steven D. Bruner, and Andrew D. Hanson

Subject:

Subject ID:SU001106
Subject Type:Plant
Subject Species:Arabidopsis thaliana
Taxonomy ID:3702
Age Or Age Range:3-4 weeks

Factors:

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

mb_sample_id local_sample_id Genotype Treatment Tissue Type
SA071837A4_38Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071838A3_37Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071839A5_39Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071840A6_40Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071841A2_36Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071842A1_35Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071843E1_25Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071844E5_29Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071845E4_28Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071846E3_27Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071847E2_26Columbai-O_WT 1/2MS-media_3weeks Whole Plant
SA071848C5_17Columbai-O_WT 1/2MS-media_4weeks Whole Plant
SA071849C4_16Columbai-O_WT 1/2MS-media_4weeks Whole Plant
SA071850C6_18Columbai-O_WT 1/2MS-media_4weeks Whole Plant
SA071851C3_15Columbai-O_WT 1/2MS-media_4weeks Whole Plant
SA071852C1_13Columbai-O_WT 1/2MS-media_4weeks Whole Plant
SA071853C2_14Columbai-O_WT 1/2MS-media_4weeks Whole Plant
SA071854A6_6Columbai-O_WT SoilGrown_4weeks Leaves
SA071855A1_1Columbai-O_WT SoilGrown_4weeks Leaves
SA071856A5_5Columbai-O_WT SoilGrown_4weeks Leaves
SA071857A3_3Columbai-O_WT SoilGrown_4weeks Leaves
SA071858A2_2Columbai-O_WT SoilGrown_4weeks Leaves
SA071859A4_4Columbai-O_WT SoilGrown_4weeks Leaves
SA071860F3_32SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071861F5_34SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071862F4_33SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071863F1_30SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071864F2_31SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071865B6_46SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071866B1_41SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071867B2_42SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071868B3_43SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071869B4_44SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071870B5_45SALK_108849C_KO 1/2MS-media_3weeks Whole Plant
SA071871D6_24SALK_108849C_KO 1/2MS-media_4weeks Whole Plant
SA071872D5_23SALK_108849C_KO 1/2MS-media_4weeks Whole Plant
SA071873D1_19SALK_108849C_KO 1/2MS-media_4weeks Whole Plant
SA071874D2_20SALK_108849C_KO 1/2MS-media_4weeks Whole Plant
SA071875D3_21SALK_108849C_KO 1/2MS-media_4weeks Whole Plant
SA071876D4_22SALK_108849C_KO 1/2MS-media_4weeks Whole Plant
SA071877B2_8SALK_108849C_KO SoilGrown_4weeks Leaves
SA071878B1_7SALK_108849C_KO SoilGrown_4weeks Leaves
SA071879B3_9SALK_108849C_KO SoilGrown_4weeks Leaves
SA071880B6_12SALK_108849C_KO SoilGrown_4weeks Leaves
SA071881B5_11SALK_108849C_KO SoilGrown_4weeks Leaves
SA071882B4_10SALK_108849C_KO SoilGrown_4weeks Leaves
Showing results 1 to 46 of 46

Collection:

Collection ID:CO001100
Collection Summary:For metabolic analysis, samples were immediately frozen in liquid nitrogen, powdered, lyophilized, and stored at -80°C until analysis.
Sample Type:Plant

Treatment:

Treatment ID:TR001120
Treatment Summary:Plants of either wild type or knockout genotype were grown on 1/2 MS media for 3-4 weeks, or grown on soil for 4 weeks.
Plant Light Period:12:12h

Sample Preparation:

Sampleprep ID:SP001113
Sampleprep Summary:Samples (10 mg lyophilized plant tissue) were extracted using biphasic extraction technique adapted from Matyash et al. 2008. In summary 225 µL LC-MS grade methanol was added to lyophilized plant tissue in 2 mL Eppendorf tube, vortexed for 10 seconds, followed by addition of 750 µL methyl tert-butyl ether (MTBE). Each sample was then vortexed for 10 seconds, shook on orbital shaker at maximum speed for six minutes, followed by addition of 188 µL LC-MS grade water. Finally, each sample was vortexed for 10 seconds and centrifuged for 2 minutes at 14,000 rpm. The resultant two-phase extract was aliquoted into four clean 1.5 mL Eppendorf tubes. The result was two 350 µL aliquots of MTBE phase (top), and two 110 µL aliquots of methanol/water phase (bottom). Extraction was carried out at 4°C. All extract tubes were dried under vacuum and frozen at -80°C until LC-MS/MS analysis. Lipidomics analysis followed methods of Cajka et al. 2017. Briefly, extract from one aliquot of MTBE phase was resuspended in 110 µL methanol/toluene (9:1, v/v), vortexed for 10 seconds, centrifuged for 2 minutes at 14,000 rpm, transferred to HPLC vial, and stored at 4°C until LC-MS/MS analysis. Gradient, internal standards, mobile phases, and data collection methods were identical to Cajka et al. 2017. Sample injection volume was 3 µL on a Waters Acquity UPLC CSH C18 column (100mm x 2.1mm, 1.7 μm particle size). A Thermo Vanquish Focused UHPLC system coupled to Thermo Q Exactive HF mass spectrometer was used for all untargeted analysis. Untargeted polar metabolomics analysis followed methods of Niehaus et al. 2018. Briefly, one aliquot of methanol/water phase extract was resuspended in 100 µL acetonitrile/water (4:1 v/v) with internal standards including 5 µg/ml Val-Tyr-Val. Analysis was carried out using Waters Acquity UPLC BEH Amide (150mm x 2.1 mm id, 1.7 μm particle size) column with identical mobile phase, gradient, injection volume, and data collection to Niehaus et al. 2018.

Combined analysis:

Analysis ID AN001736 AN001737 AN001738
Analysis type MS MS MS
Chromatography type HILIC HILIC Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish Thermo Vanquish
Column Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
MS Type ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE NEGATIVE POSITIVE
Units AU AU AU

Chromatography:

Chromatography ID:CH001228
Chromatography Summary:HILIC chromatography 17 minute run. Positive and Negative mode.
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
Column Temperature:45
Flow Gradient:The gradient started at 100% B for 2 minutes, then brought to 70% B by 7.7 minutes, 40% B by 9.5 minutes, back to 100% B by 12.75 minutes and held at 100% B until 17 minutes.
Flow Rate:0.4 mL/min
Internal Standard:5ug Val-Tyr-Val+
Sample Injection:5uL
Solvent A:water with 10 mM ammonium formate, and 0.125% formic acid
Solvent B:was LC-MS grade 95:5 v/v acetonitrile/water with 10 mM ammonium formate and 0.125% formic acid
Washing Buffer:Water:ACN 50:50
Target Sample Temperature:4
Chromatography Type:HILIC
  
Chromatography ID:CH001229
Chromatography Summary:CSH C18 lipidomics run. Positive mode. As in Cajka et al. 2017 "Validating Quantitative untargeted lipidomics across nine liquid chromatography-High-Resolution Mass spectrometry platforms"
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
Column Temperature:65
Flow Gradient:: 0 min 15% (B); 0−2 min 30% (B); 2−2.5 min 48% (B); 2.5−11 min 82% (B); 11−11.5 min 99% (B); 11.5−12 min 99% (B); 12−12.1 min 15% (B); and 12.1−15 min 15% (B)
Flow Rate:0.6 mL/min
Internal Standard:[LPE(17:1), LPC(17:0), PC(12:0/13:0), PE(17:0/17:0), PG(17:0/17:0), d7-cholesterol, SM(d18:1/17:0), Cer(d18:1/17:0), sphingosine (d17:1), DG(12:0/12:0/0:0), DG(18:1/2:0/0:0), and d5-TG- (17:0/17:1/17:0)]
Sample Injection:3uL
Solvent A:(A) 60:40 (v/v) acetonitrile:water with ammonium formate (10 mM) and formic acid (0.1%)
Solvent B:(B) 90:10 (v/v) isopropanol:acetonitrile with ammonium formate (10 mM) and formic acid (0.1%)
Washing Buffer:IPA
Target Sample Temperature:4
Chromatography Type:Reversed phase

MS:

MS ID:MS001605
Analysis ID:AN001736
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:POSITIVE
Collision Energy:NCE 20.30.40
Ionization:Positive
Scan Range Moverz:60-900
Scanning Cycle:Top 4 ions selected for MS/MS
  
MS ID:MS001606
Analysis ID:AN001737
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:NEGATIVE
Collision Energy:NCE 20.30.40
Ionization:Negative
Scan Range Moverz:60-900
Scanning Cycle:Top 4 ions selected for MS/MS
  
MS ID:MS001607
Analysis ID:AN001738
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:POSITIVE
Collision Energy:NCE 20.30.40
Ionization:Positive
Scan Range Moverz:120-1200
Scanning Cycle:Top 4 ions selected for MS/MS
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