#METABOLOMICS WORKBENCH skambhampati_20220721_095821_mwtab.txt DATATRACK_ID:3359 STUDY_ID:ST002240 ANALYSIS_ID:AN003657 PROJECT_ID:PR001429
VERSION             	1
CREATED_ON             	July 21, 2022, 10:57 am
#PROJECT
PR:PROJECT_TITLE                 	Using stable isotopes and mass spectrometry to elucidate the dynamics of
PR:PROJECT_TITLE                 	metabolic pathways
PR:PROJECT_TYPE                  	Stable Isotope Enriched Lipidomics
PR:PROJECT_SUMMARY               	Data analysis and mass spectrometry tools have advanced significantly in the
PR:PROJECT_SUMMARY               	last decade. This ongoing revolution has elevated the status of analytical
PR:PROJECT_SUMMARY               	chemistry within the big-data omics era. High resolution mass spectrometers
PR:PROJECT_SUMMARY               	(HRMS) can now distinguish different metabolites with mass to charge ratios
PR:PROJECT_SUMMARY               	(i.e. m/z) that differ by 0.01 Da or less. This unprecedented level of
PR:PROJECT_SUMMARY               	resolution not only enables identification of previously unknown compounds but
PR:PROJECT_SUMMARY               	also presents an opportunity to establish active metabolic pathways through
PR:PROJECT_SUMMARY               	quantification of isotope enrichment. Studies with stable isotope tracers
PR:PROJECT_SUMMARY               	continue to contribute to our knowledge of biological pathways in human, plant
PR:PROJECT_SUMMARY               	and bacterial species, however most current studies have been based on targeted
PR:PROJECT_SUMMARY               	analyses. The capacity of HRMS to resolve near-overlapping isotopologues and
PR:PROJECT_SUMMARY               	identify compounds with high mass precision presents a strategy to assess
PR:PROJECT_SUMMARY               	‘active’ pathways de novo from data generated in an untargeted way, that is
PR:PROJECT_SUMMARY               	blind to the metabolic network and therefore unbiased. Currently, identifying
PR:PROJECT_SUMMARY               	metabolic features, enriched with stable isotopes, at an ‘omics’ level
PR:PROJECT_SUMMARY               	remains an experimental bottleneck, limiting our capacity to understand
PR:PROJECT_SUMMARY               	biological network operation at the metabolic level. We developed data analysis
PR:PROJECT_SUMMARY               	tools that: i) use labeling information and exact mass to determine the
PR:PROJECT_SUMMARY               	elemental composition of each isotopically enriched ion, ii) apply
PR:PROJECT_SUMMARY               	correlation-based approaches to cluster metabolite peaks with similar patterns
PR:PROJECT_SUMMARY               	of isotopic labels and, iii) leverage this information to build directed
PR:PROJECT_SUMMARY               	metabolic networks de novo. Using Camelina sativa, an emerging oilseed model, we
PR:PROJECT_SUMMARY               	demonstrate the power of stable isotope labeling in combination with imaging and
PR:PROJECT_SUMMARY               	HRMS to reconstruct lipid metabolic networks in developing seeds and are
PR:PROJECT_SUMMARY               	currently addressing questions about lipid and central metabolism. Tools
PR:PROJECT_SUMMARY               	developed in this study will have a broader application to assess context
PR:PROJECT_SUMMARY               	specific operation of metabolic pathways.
PR:INSTITUTE                     	Donald Danforth Plant Science Center
PR:DEPARTMENT                    	Allen/USDA lab
PR:LABORATORY                    	Allen lab
PR:LAST_NAME                     	Shrikaar
PR:FIRST_NAME                    	Kambhampati
PR:ADDRESS                       	975 North Warson road
PR:EMAIL                         	skambhampati@danforthcenter.org
PR:PHONE                         	3144025550
PR:FUNDING_SOURCE                	NIH, USDA-ARS
#STUDY
ST:STUDY_TITLE                   	Use of HRMS and Dual Isotope Labels to Resolve Difficult-to Measure Fluxes
ST:STUDY_TYPE                    	Stable isotope enriched Metabolomics
ST:STUDY_SUMMARY                 	Data analysis and mass spectrometry tools have advanced significantly in the
ST:STUDY_SUMMARY                 	last decade. This ongoing revolution has elevated the status of analytical
ST:STUDY_SUMMARY                 	chemistry within the big-data omics era. High resolution mass spectrometers
ST:STUDY_SUMMARY                 	(HRMS) can now distinguish different metabolites with mass to charge ratios
ST:STUDY_SUMMARY                 	(i.e. m/z) that differ by 0.01 Da or less. This unprecedented level of
ST:STUDY_SUMMARY                 	resolution not only enables identification of previously unknown compounds but
ST:STUDY_SUMMARY                 	also presents an opportunity to establish active metabolic pathways through
ST:STUDY_SUMMARY                 	quantification of isotope enrichment. Studies with stable isotope tracers
ST:STUDY_SUMMARY                 	continue to contribute to our knowledge of biological pathways in human, plant
ST:STUDY_SUMMARY                 	and bacterial species, however most current studies have been based on targeted
ST:STUDY_SUMMARY                 	analyses. The capacity of HRMS to resolve near-overlapping isotopologues and
ST:STUDY_SUMMARY                 	identify compounds with high mass precision presents a strategy to assess
ST:STUDY_SUMMARY                 	‘active’ pathways de novo from data generated in an untargeted way, that is
ST:STUDY_SUMMARY                 	blind to the metabolic network and therefore unbiased. Currently, identifying
ST:STUDY_SUMMARY                 	metabolic features, enriched with stable isotopes, at an ‘omics’ level
ST:STUDY_SUMMARY                 	remains an experimental bottleneck, limiting our capacity to understand
ST:STUDY_SUMMARY                 	biological network operation at the metabolic level. We developed data analysis
ST:STUDY_SUMMARY                 	tools that: i) use labeling information and exact mass to determine the
ST:STUDY_SUMMARY                 	elemental composition of each isotopically enriched ion, ii) apply
ST:STUDY_SUMMARY                 	correlation-based approaches to cluster metabolite peaks with similar patterns
ST:STUDY_SUMMARY                 	of isotopic labels and, iii) leverage this information to build directed
ST:STUDY_SUMMARY                 	metabolic networks de novo. Using Camelina sativa, an emerging oilseed model, we
ST:STUDY_SUMMARY                 	demonstrate the power of stable isotope labeling in combination with imaging and
ST:STUDY_SUMMARY                 	HRMS to reconstruct lipid metabolic networks in developing seeds and are
ST:STUDY_SUMMARY                 	currently addressing questions about lipid and central metabolism. Tools
ST:STUDY_SUMMARY                 	developed in this study will have a broader application to assess context
ST:STUDY_SUMMARY                 	specific operation of metabolic pathways.
ST:INSTITUTE                     	Donald Danforth Plant Science Center
ST:DEPARTMENT                    	Allen/USDA lab
ST:LABORATORY                    	Allen Lab
ST:LAST_NAME                     	Shrikaar
ST:FIRST_NAME                    	Kambhampati
ST:ADDRESS                       	975 North Warson road
ST:EMAIL                         	skambhampati@danforthcenter.org
ST:PHONE                         	3144025550
#SUBJECT
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Arabidopsis thaliana
SU:TAXONOMY_ID                   	3702
SU:AGE_OR_AGE_RANGE              	10 day old seedlings
SU:SPECIES_GROUP                 	Roots
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	0_GAT_1-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):0	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=0_GAT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	0_GAT_1-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):0	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=0_GAT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	0_GAT_2-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):0	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=0_GAT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	0_GAT_2-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):0	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=0_GAT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	0_GAT_3-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):0	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=0_GAT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	0_GAT_3-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):0	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=0_GAT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	0_GAT_4-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):0	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=0_GAT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	0_GAT_4-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):0	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=0_GAT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	0_WT_1-pos	Tissue type:Wildtype (Col 0) | Time (hours):0	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=0_WT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	0_WT_1-neg	Tissue type:Wildtype (Col 0) | Time (hours):0	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=0_WT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	0_WT_2-pos	Tissue type:Wildtype (Col 0) | Time (hours):0	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=0_WT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	0_WT_2-neg	Tissue type:Wildtype (Col 0) | Time (hours):0	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=0_WT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	0_WT_3-pos	Tissue type:Wildtype (Col 0) | Time (hours):0	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=0_WT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	0_WT_3-neg	Tissue type:Wildtype (Col 0) | Time (hours):0	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=0_WT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	0_WT_4-pos	Tissue type:Wildtype (Col 0) | Time (hours):0	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=0_WT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	0_WT_4-neg	Tissue type:Wildtype (Col 0) | Time (hours):0	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=0_WT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	2_GAT_1-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):2	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=2_GAT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	2_GAT_1-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):2	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=2_GAT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	2_GAT_2-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):2	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=2_GAT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	2_GAT_2-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):2	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=2_GAT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	2_GAT_3-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):2	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=2_GAT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	2_GAT_3-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):2	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=2_GAT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	2_GAT_4-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):2	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=2_GAT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	2_GAT_4-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):2	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=2_GAT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	2_WT_1-pos	Tissue type:Wildtype (Col 0) | Time (hours):2	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=2_WT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	2_WT_1-neg	Tissue type:Wildtype (Col 0) | Time (hours):2	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=2_WT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	2_WT_2-pos	Tissue type:Wildtype (Col 0) | Time (hours):2	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=2_WT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	2_WT_2-neg	Tissue type:Wildtype (Col 0) | Time (hours):2	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=2_WT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	2_WT_3-pos	Tissue type:Wildtype (Col 0) | Time (hours):2	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=2_WT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	2_WT_3-neg	Tissue type:Wildtype (Col 0) | Time (hours):2	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=2_WT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	2_WT_4-pos	Tissue type:Wildtype (Col 0) | Time (hours):2	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=2_WT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	2_WT_4-neg	Tissue type:Wildtype (Col 0) | Time (hours):2	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=2_WT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	4_GAT_1-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):4	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=4_GAT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	4_GAT_1-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):4	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=4_GAT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	4_GAT_2-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):4	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=4_GAT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	4_GAT_2-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):4	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=4_GAT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	4_GAT_3-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):4	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=4_GAT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	4_GAT_3-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):4	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=4_GAT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	4_GAT_4-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):4	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=4_GAT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	4_GAT_4-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):4	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=4_GAT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	4_WT_1-pos	Tissue type:Wildtype (Col 0) | Time (hours):4	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=4_WT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	4_WT_1-neg	Tissue type:Wildtype (Col 0) | Time (hours):4	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=4_WT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	4_WT_2-pos	Tissue type:Wildtype (Col 0) | Time (hours):4	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=4_WT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	4_WT_2-neg	Tissue type:Wildtype (Col 0) | Time (hours):4	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=4_WT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	4_WT_3-pos	Tissue type:Wildtype (Col 0) | Time (hours):4	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=4_WT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	4_WT_3-neg	Tissue type:Wildtype (Col 0) | Time (hours):4	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=4_WT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	4_WT_4-pos	Tissue type:Wildtype (Col 0) | Time (hours):4	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=4_WT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	4_WT_4-neg	Tissue type:Wildtype (Col 0) | Time (hours):4	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=4_WT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	6_GAT_1-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):6	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=6_GAT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	6_GAT_1-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):6	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=6_GAT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	6_GAT_2-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):6	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=6_GAT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	6_GAT_2-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):6	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=6_GAT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	6_GAT_3-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):6	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=6_GAT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	6_GAT_3-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):6	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=6_GAT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	6_GAT_4-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):6	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=6_GAT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	6_GAT_4-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):6	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=6_GAT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	6_WT_1-pos	Tissue type:Wildtype (Col 0) | Time (hours):6	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=6_WT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	6_WT_1-neg	Tissue type:Wildtype (Col 0) | Time (hours):6	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=6_WT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	6_WT_2-pos	Tissue type:Wildtype (Col 0) | Time (hours):6	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=6_WT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	6_WT_2-neg	Tissue type:Wildtype (Col 0) | Time (hours):6	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=6_WT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	6_WT_3-pos	Tissue type:Wildtype (Col 0) | Time (hours):6	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=6_WT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	6_WT_3-neg	Tissue type:Wildtype (Col 0) | Time (hours):6	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=6_WT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	6_WT_4-pos	Tissue type:Wildtype (Col 0) | Time (hours):6	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=6_WT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	6_WT_4-neg	Tissue type:Wildtype (Col 0) | Time (hours):6	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=6_WT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	8_GAT_1-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):8	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=8_GAT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	8_GAT_1-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):8	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=8_GAT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	8_GAT_2-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):8	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=8_GAT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	8_GAT_2-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):8	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=8_GAT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	8_GAT_3-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):8	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=8_GAT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	8_GAT_3-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):8	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=8_GAT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	8_GAT_4-pos	Tissue type:gat1_2.1 (mutant) | Time (hours):8	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=8_GAT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	8_GAT_4-neg	Tissue type:gat1_2.1 (mutant) | Time (hours):8	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=8_GAT_4-neg
SUBJECT_SAMPLE_FACTORS           	-	8_WT_1-pos	Tissue type:Wildtype (Col 0) | Time (hours):8	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=8_WT_1-pos
SUBJECT_SAMPLE_FACTORS           	-	8_WT_1-neg	Tissue type:Wildtype (Col 0) | Time (hours):8	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=8_WT_1-neg
SUBJECT_SAMPLE_FACTORS           	-	8_WT_2-pos	Tissue type:Wildtype (Col 0) | Time (hours):8	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=8_WT_2-pos
SUBJECT_SAMPLE_FACTORS           	-	8_WT_2-neg	Tissue type:Wildtype (Col 0) | Time (hours):8	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=8_WT_2-neg
SUBJECT_SAMPLE_FACTORS           	-	8_WT_3-pos	Tissue type:Wildtype (Col 0) | Time (hours):8	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=8_WT_3-pos
SUBJECT_SAMPLE_FACTORS           	-	8_WT_3-neg	Tissue type:Wildtype (Col 0) | Time (hours):8	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=8_WT_3-neg
SUBJECT_SAMPLE_FACTORS           	-	8_WT_4-pos	Tissue type:Wildtype (Col 0) | Time (hours):8	Chromatography=HILIC; Polarity=Positive; RAW_FILE_NAME=8_WT_4-pos
SUBJECT_SAMPLE_FACTORS           	-	8_WT_4-neg	Tissue type:Wildtype (Col 0) | Time (hours):8	Chromatography=HILIC; Polarity=Negative; RAW_FILE_NAME=8_WT_4-neg
#COLLECTION
CO:COLLECTION_SUMMARY            	For the metabolomics study using dual-isotope labeling, wildtype Arabidopsis
CO:COLLECTION_SUMMARY            	ecotype Columbia seeds were grown on vertical plates at 22°C under continuous
CO:COLLECTION_SUMMARY            	light (ca. 70 µmol m-2 s-1), on a defined nutrient medium previously
CO:COLLECTION_SUMMARY            	described11. The medium consisted of 10 mM potassium phosphate (pH 6.5), 5 mM
CO:COLLECTION_SUMMARY            	KNO3, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNaEDTA, micronutrients (50 mM H3BO3, 12
CO:COLLECTION_SUMMARY            	mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar.
CO:COLLECTION_SUMMARY            	Ten-day old seedlings were transferred to plates containing the same medium,
CO:COLLECTION_SUMMARY            	except the nitrogen source was replaced with 10 mM [13C5,15N2]glutamine. Root
CO:COLLECTION_SUMMARY            	tissue was excised after exposure to medium containing labeled glutamine for 2,
CO:COLLECTION_SUMMARY            	4, 6 and 8h to represent time course incorporation of carbon and nitrogen into
CO:COLLECTION_SUMMARY            	metabolism. Untreated roots were used as unlabeled (0h) controls. Each plate
CO:COLLECTION_SUMMARY            	yielded ~100 mg of root tissue and served as a single replicate. Four replicates
CO:COLLECTION_SUMMARY            	per sample type were collected and flash frozen using liquid N2 for total
CO:COLLECTION_SUMMARY            	metabolite extraction.
CO:SAMPLE_TYPE                   	Plant
CO:COLLECTION_METHOD             	Flash frozen in Liquid N2
CO:COLLECTION_LOCATION           	Donald Danforth Plant Science Center
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	For the metabolomics study using dual-isotope labeling, wildtype Arabidopsis
TR:TREATMENT_SUMMARY             	ecotype Columbia seeds were grown on vertical plates at 22°C under continuous
TR:TREATMENT_SUMMARY             	light (ca. 70 µmol m-2 s-1), on a defined nutrient medium previously
TR:TREATMENT_SUMMARY             	described11. The medium consisted of 10 mM potassium phosphate (pH 6.5), 5 mM
TR:TREATMENT_SUMMARY             	KNO3, 2 mM MgSO4, 1 mM CaCl2, 0.1 mM FeNaEDTA, micronutrients (50 mM H3BO3, 12
TR:TREATMENT_SUMMARY             	mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar.
TR:TREATMENT_SUMMARY             	Ten-day old seedlings were transferred to plates containing the same medium,
TR:TREATMENT_SUMMARY             	except the nitrogen source was replaced with 10 mM [13C5,15N2]glutamine. Root
TR:TREATMENT_SUMMARY             	tissue was excised after exposure to medium containing labeled glutamine for 2,
TR:TREATMENT_SUMMARY             	4, 6 and 8h to represent time course incorporation of carbon and nitrogen into
TR:TREATMENT_SUMMARY             	metabolism. Untreated roots were used as unlabeled (0h) controls. Each plate
TR:TREATMENT_SUMMARY             	yielded ~100 mg of root tissue and served as a single replicate. Four replicates
TR:TREATMENT_SUMMARY             	per sample type were collected and flash frozen using liquid N2 for total
TR:TREATMENT_SUMMARY             	metabolite extraction.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Frozen Arabidopsis root tissue was homogenized using a tissue lyser, and
SP:SAMPLEPREP_SUMMARY            	extraction was carried out using 1 mL of 4:1 methanol: water (v/v) with
SP:SAMPLEPREP_SUMMARY            	incubation in an ultra-sonication bath for 30 min followed by shaking for 30 min
SP:SAMPLEPREP_SUMMARY            	at 4°C. The mixture was then centrifuged at 21,000 x g for 10 min at 4°C;
SP:SAMPLEPREP_SUMMARY            	supernatant was transferred into fresh tubes and evaporated to dryness using a
SP:SAMPLEPREP_SUMMARY            	speedvac centrifuge at ambient temperature. Dried residue was re-suspended in
SP:SAMPLEPREP_SUMMARY            	200 µL of 1:1 methanol: water (v/v), filtered using 0.2 µm PTFE micro
SP:SAMPLEPREP_SUMMARY            	centrifuge filters and transferred to glass vials for HILIC-HRMS runs.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACTION_METHOD             	4:1 Methanol:Water
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Chromatographic separation using HILIC was achieved using an Agilent 1290
CH:CHROMATOGRAPHY_SUMMARY        	Infinity II UHPLC system equipped with a SeQuant® ZIC®-HILIC (100 x 2.1 x 3.5
CH:CHROMATOGRAPHY_SUMMARY        	µm) column (EMD Millipore, Burlington, MA). Mobile phases A and B were
CH:CHROMATOGRAPHY_SUMMARY        	comprised of 5 mM ammonium acetate (pH 4.0) in water and 90% acetonitrile with
CH:CHROMATOGRAPHY_SUMMARY        	0.1 % acetic acid, respectively. A flow rate of 0.3 mL min-1 was used to elute
CH:CHROMATOGRAPHY_SUMMARY        	compounds with the following gradient: 87% B for 5 minutes, decreased to 55% B
CH:CHROMATOGRAPHY_SUMMARY        	over the next 8 minutes and held for 2.5 minutes before returning to 87% and
CH:CHROMATOGRAPHY_SUMMARY        	equilibrating the column for 3 minutes.
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity II
CH:COLUMN_NAME                   	SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)
CH:FLOW_RATE                     	0.3 mL min-1
CH:COLUMN_TEMPERATURE            	40
CH:SOLVENT_A                     	5 mM ammonium acetate (pH 4.0) in water
CH:SOLVENT_B                     	90% acetonitrile with 0.1 % acetic acid
CH:INTERNAL_STANDARD             	Equisplash
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The heated electrospray ionization (HESI) conditions used were as follows; spray
MS:MS_COMMENTS                   	voltage, 3.9 kV (ESI+), 3.5 kV (ESI-); capillary temperature, 250 °C; probe
MS:MS_COMMENTS                   	heater temperature, 450 °C; sheath gas, 30 arbitrary units; auxiliary gas, 8
MS:MS_COMMENTS                   	arbitrary units; and S-Lens RF level, 60%. Full MS data were collected using a
MS:MS_COMMENTS                   	Q-Exactive Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific) in
MS:MS_COMMENTS                   	both positive and negative ionization mode separately from mass ranges 75-1100
MS:MS_COMMENTS                   	m/z and 65-900 m/z, respectively, at 140,000 resolution. The automatic gain
MS:MS_COMMENTS                   	control (AGC) was set to 3 x 106 and maximum injection time (IT) used was 524
MS:MS_COMMENTS                   	ms.
MS:MS_RESULTS_FILE               	ST002240_AN003657_Results.txt	UNITS:Intensity	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END