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.

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

Study ID	ST001062
Study Title	Arabidopsis Nit1 knockout metabolomics
Study Summary	Glutathione (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 Name	Folz
First Name	Jacob
Address	451 Health Sciences Dr., Davis, CA, 95616
Email	jfolz@ucdavis.edu
Phone	7155636311
Submit Date	2018-09-24
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2019-03-06
Release Version	1

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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
SA071837	A4_38	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071838	A3_37	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071839	A5_39	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071840	A6_40	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071841	A2_36	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071842	A1_35	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071843	E1_25	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071844	E5_29	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071845	E4_28	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071846	E3_27	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071847	E2_26	Columbai-O_WT	1/2MS-media_3weeks	Whole Plant
SA071848	C5_17	Columbai-O_WT	1/2MS-media_4weeks	Whole Plant
SA071849	C4_16	Columbai-O_WT	1/2MS-media_4weeks	Whole Plant
SA071850	C6_18	Columbai-O_WT	1/2MS-media_4weeks	Whole Plant
SA071851	C3_15	Columbai-O_WT	1/2MS-media_4weeks	Whole Plant
SA071852	C1_13	Columbai-O_WT	1/2MS-media_4weeks	Whole Plant
SA071853	C2_14	Columbai-O_WT	1/2MS-media_4weeks	Whole Plant
SA071854	A6_6	Columbai-O_WT	SoilGrown_4weeks	Leaves
SA071855	A1_1	Columbai-O_WT	SoilGrown_4weeks	Leaves
SA071856	A5_5	Columbai-O_WT	SoilGrown_4weeks	Leaves
SA071857	A3_3	Columbai-O_WT	SoilGrown_4weeks	Leaves
SA071858	A2_2	Columbai-O_WT	SoilGrown_4weeks	Leaves
SA071859	A4_4	Columbai-O_WT	SoilGrown_4weeks	Leaves
SA071860	F3_32	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071861	F5_34	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071862	F4_33	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071863	F1_30	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071864	F2_31	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071865	B6_46	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071866	B1_41	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071867	B2_42	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071868	B3_43	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071869	B4_44	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071870	B5_45	SALK_108849C_KO	1/2MS-media_3weeks	Whole Plant
SA071871	D6_24	SALK_108849C_KO	1/2MS-media_4weeks	Whole Plant
SA071872	D5_23	SALK_108849C_KO	1/2MS-media_4weeks	Whole Plant
SA071873	D1_19	SALK_108849C_KO	1/2MS-media_4weeks	Whole Plant
SA071874	D2_20	SALK_108849C_KO	1/2MS-media_4weeks	Whole Plant
SA071875	D3_21	SALK_108849C_KO	1/2MS-media_4weeks	Whole Plant
SA071876	D4_22	SALK_108849C_KO	1/2MS-media_4weeks	Whole Plant
SA071877	B2_8	SALK_108849C_KO	SoilGrown_4weeks	Leaves
SA071878	B1_7	SALK_108849C_KO	SoilGrown_4weeks	Leaves
SA071879	B3_9	SALK_108849C_KO	SoilGrown_4weeks	Leaves
SA071880	B6_12	SALK_108849C_KO	SoilGrown_4weeks	Leaves
SA071881	B5_11	SALK_108849C_KO	SoilGrown_4weeks	Leaves
SA071882	B4_10	SALK_108849C_KO	SoilGrown_4weeks	Leaves

Showing results 1 to 46 of 46

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.

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:	100% water; 0.125% formic acid; 10 mM ammonium formate
Solvent B:	95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate
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:	60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
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