{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001062","ANALYSIS_ID":"AN001738","VERSION":"1","CREATED_ON":"September 24, 2018, 3:41 pm"},

"PROJECT":{"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"},

"STUDY":{"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"},

"SUBJECT":{"SUBJECT_TYPE":"Plant","SUBJECT_SPECIES":"Arabidopsis thaliana","TAXONOMY_ID":"3702","AGE_OR_AGE_RANGE":"3-4 weeks"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"A1_1",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"A2_2",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"A3_3",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"A4_4",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"A5_5",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"A6_6",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"B1_7",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"B2_8",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"B3_9",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"B4_10",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"B5_11",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"B6_12",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"SoilGrown_4weeks","Tissue Type":"Leaves"}
},
{
"Subject ID":"-",
"Sample ID":"C1_13",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"C2_14",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"C3_15",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"C4_16",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"C5_17",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"C6_18",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"D1_19",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"D2_20",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"D3_21",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"D4_22",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"D5_23",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"D6_24",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_4weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"E1_25",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"E2_26",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"E3_27",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"E4_28",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"E5_29",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"F1_30",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"F2_31",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"F3_32",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"F4_33",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"F5_34",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"A1_35",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"A2_36",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"A3_37",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"A4_38",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"A5_39",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"A6_40",
"Factors":{"Genotype":"Columbai-O_WT","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"B1_41",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"B2_42",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"B3_43",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"B4_44",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"B5_45",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
},
{
"Subject ID":"-",
"Sample ID":"B6_46",
"Factors":{"Genotype":"SALK_108849C_KO","Treatment":"1/2MS-media_3weeks","Tissue Type":"Whole Plant"}
}
],
"COLLECTION":{"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_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"},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"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","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)","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","COLUMN_TEMPERATURE":"65","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%)","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","WASHING_BUFFER":"IPA","TARGET_SAMPLE_TEMPERATURE":"4"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Fiehn Lab","OPERATOR_NAME":"Jacob Folz","DETECTOR_TYPE":"Orbitrap","ACQUISITION_DATE":"04_19_2018","DATA_FORMAT":"Data Dependent Acquisition"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","ACQUISITION_DATE":"04_19_2018","COLLISION_ENERGY":"NCE 20.30.40","IONIZATION":"Positive","SCAN_RANGE_MOVERZ":"120-1200","SCANNING_CYCLE":"Top 4 ions selected for MS/MS"},

"MS_METABOLITE_DATA":{
"Units":"AU",

"Data":[{"Metabolite":"AC(14:2)","A1_1":"1236159","A2_2":"933097","A3_3":"1058629","A4_4":"535416","A5_5":"1539612","A6_6":"179820","B1_7":"405625","B2_8":"779665","B3_9":"416120","B4_10":"891695","B5_11":"182693","B6_12":"1038681","C1_13":"1467541","C2_14":"1869242","C3_15":"2196340","C4_16":"1103980","C5_17":"1260722","C6_18":"2530302","D1_19":"1368983","D2_20":"818974","D3_21":"1388495","D4_22":"1504227","D5_23":"2417347","D6_24":"1199269","E1_25":"991630","E2_26":"3158979","E3_27":"568477","E4_28":"649404","E5_29":"1543602","F1_30":"5006658","F2_31":"583127","F3_32":"2608223","F4_33":"638001","F5_34":"1071043","A1_35":"2610278","A2_36":"1102118","A3_37":"1782858","A4_38":"1323769","A5_39":"2688203","A6_40":"1845817","B1_41":"2174312","B2_42":"3782601","B3_43":"2080907","B4_44":"1666811","B5_45":"1254814","B6_46":"612070"},{"Metabolite":"AcylGlcADG 50:2; AcylGlcADG 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m/z":"1012.9283","rtimes(mins)":"11.84","Adduct":"[M+NH4]+","InchiKey":"AHXIACCOHSVLRP-ULZSGHTGSA-N"}]
}

}