#METABOLOMICS WORKBENCH skambhampati_20220721_075626 DATATRACK_ID:3358 STUDY_ID:ST002239 ANALYSIS_ID:AN003656
VERSION                          	1
CREATED_ON                       	08-10-2022
#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, St. Louis, MO 63132
PR:EMAIL                         	skambhampati@danforthcenter.org
PR:PHONE                         	3144025550
PR:FUNDING_SOURCE                	NIH, USDA-ARS
PR:DOI                           	http://dx.doi.org/10.21228/M80X3B
#STUDY
ST:STUDY_TITLE                   	Insights into plant lipid metabolism using stable isotopes and high resolution
ST:STUDY_TITLE                   	mass spectrometry
ST:STUDY_TYPE                    	Stable isotope enriched lipidomics
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
ST:SUBMIT_DATE                   	2022-07-21
#SUBJECT
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Camelina Sativa
SU:AGE_OR_AGE_RANGE              	10 days after fertilization
SU:SPECIES_GROUP                 	Developing seeds
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	0_Cotyledon_1-neg	Tissue type:Cotyledon | Time (hours):0	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_Cotyledon_1-neg
SUBJECT_SAMPLE_FACTORS           	-	0_Cotyledon_1-pos	Tissue type:Cotyledon | Time (hours):0	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_Cotyledon_1-pos
SUBJECT_SAMPLE_FACTORS           	-	0_Cotyledon_2-neg	Tissue type:Cotyledon | Time (hours):0	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_Cotyledon_2-neg
SUBJECT_SAMPLE_FACTORS           	-	0_Cotyledon_2-pos	Tissue type:Cotyledon | Time (hours):0	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_Cotyledon_2-pos
SUBJECT_SAMPLE_FACTORS           	-	0_Cotyledon_3-neg	Tissue type:Cotyledon | Time (hours):0	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_Cotyledon_3-neg
SUBJECT_SAMPLE_FACTORS           	-	0_Cotyledon_3-pos	Tissue type:Cotyledon | Time (hours):0	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_Cotyledon_3-pos
SUBJECT_SAMPLE_FACTORS           	-	16_Cotyledon_1-neg	Tissue type:Cotyledon | Time (hours):16	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_Cotyledon_1-neg
SUBJECT_SAMPLE_FACTORS           	-	16_Cotyledon_1-pos	Tissue type:Cotyledon | Time (hours):16	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_Cotyledon_1-pos
SUBJECT_SAMPLE_FACTORS           	-	16_Cotyledon_2-neg	Tissue type:Cotyledon | Time (hours):16	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_Cotyledon_2-neg
SUBJECT_SAMPLE_FACTORS           	-	16_Cotyledon_2-pos	Tissue type:Cotyledon | Time (hours):16	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_Cotyledon_2-pos
SUBJECT_SAMPLE_FACTORS           	-	16_Cotyledon_3-neg	Tissue type:Cotyledon | Time (hours):16	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_Cotyledon_3-neg
SUBJECT_SAMPLE_FACTORS           	-	16_Cotyledon_3-pos	Tissue type:Cotyledon | Time (hours):16	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_Cotyledon_3-pos
SUBJECT_SAMPLE_FACTORS           	-	2_Cotyledon_1-neg	Tissue type:Cotyledon | Time (hours):2	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_Cotyledon_1-neg
SUBJECT_SAMPLE_FACTORS           	-	2_Cotyledon_1-pos	Tissue type:Cotyledon | Time (hours):2	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_Cotyledon_1-pos
SUBJECT_SAMPLE_FACTORS           	-	2_Cotyledon_2-neg	Tissue type:Cotyledon | Time (hours):2	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_Cotyledon_2-neg
SUBJECT_SAMPLE_FACTORS           	-	2_Cotyledon_2-pos	Tissue type:Cotyledon | Time (hours):2	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_Cotyledon_2-pos
SUBJECT_SAMPLE_FACTORS           	-	2_Cotyledon_3-neg	Tissue type:Cotyledon | Time (hours):2	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_Cotyledon_3-neg
SUBJECT_SAMPLE_FACTORS           	-	2_Cotyledon_3-pos	Tissue type:Cotyledon | Time (hours):2	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_Cotyledon_3-pos
SUBJECT_SAMPLE_FACTORS           	-	32_Cotyledon_1-neg	Tissue type:Cotyledon | Time (hours):32	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_Cotyledon_1-neg
SUBJECT_SAMPLE_FACTORS           	-	32_Cotyledon_1-pos	Tissue type:Cotyledon | Time (hours):32	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_Cotyledon_1-pos
SUBJECT_SAMPLE_FACTORS           	-	32_Cotyledon_2-neg	Tissue type:Cotyledon | Time (hours):32	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_Cotyledon_2-neg
SUBJECT_SAMPLE_FACTORS           	-	32_Cotyledon_2-pos	Tissue type:Cotyledon | Time (hours):32	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_Cotyledon_2-pos
SUBJECT_SAMPLE_FACTORS           	-	32_Cotyledon_3-neg	Tissue type:Cotyledon | Time (hours):32	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_Cotyledon_3-neg
SUBJECT_SAMPLE_FACTORS           	-	32_Cotyledon_3-pos	Tissue type:Cotyledon | Time (hours):32	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_Cotyledon_3-pos
SUBJECT_SAMPLE_FACTORS           	-	4_Cotyledon_1-neg	Tissue type:Cotyledon | Time (hours):4	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_Cotyledon_1-neg
SUBJECT_SAMPLE_FACTORS           	-	4_Cotyledon_1-pos	Tissue type:Cotyledon | Time (hours):4	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_Cotyledon_1-pos
SUBJECT_SAMPLE_FACTORS           	-	4_Cotyledon_2-neg	Tissue type:Cotyledon | Time (hours):4	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_Cotyledon_2-neg
SUBJECT_SAMPLE_FACTORS           	-	4_Cotyledon_2-pos	Tissue type:Cotyledon | Time (hours):4	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_Cotyledon_2-pos
SUBJECT_SAMPLE_FACTORS           	-	4_Cotyledon_3-neg	Tissue type:Cotyledon | Time (hours):4	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_Cotyledon_3-neg
SUBJECT_SAMPLE_FACTORS           	-	4_Cotyledon_3-pos	Tissue type:Cotyledon | Time (hours):4	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_Cotyledon_3-pos
SUBJECT_SAMPLE_FACTORS           	-	8_Cotyledon_1-neg	Tissue type:Cotyledon | Time (hours):8	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_Cotyledon_1-neg
SUBJECT_SAMPLE_FACTORS           	-	8_Cotyledon_1-pos	Tissue type:Cotyledon | Time (hours):8	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_Cotyledon_1-pos
SUBJECT_SAMPLE_FACTORS           	-	8_Cotyledon_2-neg	Tissue type:Cotyledon | Time (hours):8	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_Cotyledon_2-neg
SUBJECT_SAMPLE_FACTORS           	-	8_Cotyledon_2-pos	Tissue type:Cotyledon | Time (hours):8	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_Cotyledon_2-pos
SUBJECT_SAMPLE_FACTORS           	-	8_Cotyledon_3-neg	Tissue type:Cotyledon | Time (hours):8	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_Cotyledon_3-neg
SUBJECT_SAMPLE_FACTORS           	-	8_Cotyledon_3-pos	Tissue type:Cotyledon | Time (hours):8	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_Cotyledon_3-pos
SUBJECT_SAMPLE_FACTORS           	-	0_EA_1-neg	Tissue type:Embyo axis | Time (hours):0	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_EA_1-neg
SUBJECT_SAMPLE_FACTORS           	-	0_EA_1-pos	Tissue type:Embyo axis | Time (hours):0	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_EA_1-pos
SUBJECT_SAMPLE_FACTORS           	-	0_EA_2-neg	Tissue type:Embyo axis | Time (hours):0	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_EA_2-neg
SUBJECT_SAMPLE_FACTORS           	-	0_EA_2-pos	Tissue type:Embyo axis | Time (hours):0	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_EA_2-pos
SUBJECT_SAMPLE_FACTORS           	-	0_EA_3-neg	Tissue type:Embyo axis | Time (hours):0	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_EA_3-neg
SUBJECT_SAMPLE_FACTORS           	-	0_EA_3-pos	Tissue type:Embyo axis | Time (hours):0	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_EA_3-pos
SUBJECT_SAMPLE_FACTORS           	-	16_EA_1-neg	Tissue type:Embyo axis | Time (hours):16	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_EA_1-neg
SUBJECT_SAMPLE_FACTORS           	-	16_EA_1-pos	Tissue type:Embyo axis | Time (hours):16	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_EA_1-pos
SUBJECT_SAMPLE_FACTORS           	-	16_EA_2-neg	Tissue type:Embyo axis | Time (hours):16	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_EA_2-neg
SUBJECT_SAMPLE_FACTORS           	-	16_EA_2-pos	Tissue type:Embyo axis | Time (hours):16	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_EA_2-pos
SUBJECT_SAMPLE_FACTORS           	-	16_EA_3-neg	Tissue type:Embyo axis | Time (hours):16	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_EA_3-neg
SUBJECT_SAMPLE_FACTORS           	-	16_EA_3-pos	Tissue type:Embyo axis | Time (hours):16	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_EA_3-pos
SUBJECT_SAMPLE_FACTORS           	-	2_EA_1-neg	Tissue type:Embyo axis | Time (hours):2	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_EA_1-neg
SUBJECT_SAMPLE_FACTORS           	-	2_EA_1-pos	Tissue type:Embyo axis | Time (hours):2	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_EA_1-pos
SUBJECT_SAMPLE_FACTORS           	-	2_EA_2-neg	Tissue type:Embyo axis | Time (hours):2	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_EA_2-neg
SUBJECT_SAMPLE_FACTORS           	-	2_EA_2-pos	Tissue type:Embyo axis | Time (hours):2	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_EA_2-pos
SUBJECT_SAMPLE_FACTORS           	-	2_EA_3-neg	Tissue type:Embyo axis | Time (hours):2	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_EA_3-neg
SUBJECT_SAMPLE_FACTORS           	-	2_EA_3-pos	Tissue type:Embyo axis | Time (hours):2	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_EA_3-pos
SUBJECT_SAMPLE_FACTORS           	-	32_EA_1-neg	Tissue type:Embyo axis | Time (hours):32	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_EA_1-neg
SUBJECT_SAMPLE_FACTORS           	-	32_EA_1-pos	Tissue type:Embyo axis | Time (hours):32	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_EA_1-pos
SUBJECT_SAMPLE_FACTORS           	-	32_EA_2-neg	Tissue type:Embyo axis | Time (hours):32	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_EA_2-neg
SUBJECT_SAMPLE_FACTORS           	-	32_EA_2-pos	Tissue type:Embyo axis | Time (hours):32	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_EA_2-pos
SUBJECT_SAMPLE_FACTORS           	-	32_EA_3-neg	Tissue type:Embyo axis | Time (hours):32	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_EA_3-neg
SUBJECT_SAMPLE_FACTORS           	-	32_EA_3-pos	Tissue type:Embyo axis | Time (hours):32	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_EA_3-pos
SUBJECT_SAMPLE_FACTORS           	-	4_EA_1-neg	Tissue type:Embyo axis | Time (hours):4	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_EA_1-neg
SUBJECT_SAMPLE_FACTORS           	-	4_EA_1-pos	Tissue type:Embyo axis | Time (hours):4	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_EA_1-pos
SUBJECT_SAMPLE_FACTORS           	-	4_EA_2-neg	Tissue type:Embyo axis | Time (hours):4	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_EA_2-neg
SUBJECT_SAMPLE_FACTORS           	-	4_EA_2-pos	Tissue type:Embyo axis | Time (hours):4	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_EA_2-pos
SUBJECT_SAMPLE_FACTORS           	-	4_EA_3-neg	Tissue type:Embyo axis | Time (hours):4	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_EA_3-neg
SUBJECT_SAMPLE_FACTORS           	-	4_EA_3-pos	Tissue type:Embyo axis | Time (hours):4	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_EA_3-pos
SUBJECT_SAMPLE_FACTORS           	-	8_EA_1-neg	Tissue type:Embyo axis | Time (hours):8	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_EA_1-neg
SUBJECT_SAMPLE_FACTORS           	-	8_EA_1-pos	Tissue type:Embyo axis | Time (hours):8	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_EA_1-pos
SUBJECT_SAMPLE_FACTORS           	-	8_EA_2-neg	Tissue type:Embyo axis | Time (hours):8	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_EA_2-neg
SUBJECT_SAMPLE_FACTORS           	-	8_EA_2-pos	Tissue type:Embyo axis | Time (hours):8	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_EA_2-pos
SUBJECT_SAMPLE_FACTORS           	-	8_EA_3-neg	Tissue type:Embyo axis | Time (hours):8	Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_EA_3-neg
SUBJECT_SAMPLE_FACTORS           	-	8_EA_3-pos	Tissue type:Embyo axis | Time (hours):8	Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_EA_3-pos
#COLLECTION
CO:COLLECTION_SUMMARY            	Plant growth and culture conditions: Plants were grown in greenhouses with
CO:COLLECTION_SUMMARY            	day/night temperature maintained at 22/20°C, 40-50% relative humidity, and 16h
CO:COLLECTION_SUMMARY            	day/8h night photoperiod. Intact siliques during the seed filling growth stage
CO:COLLECTION_SUMMARY            	(15 days after fertilization) were excised and placed in sterile media
CO:COLLECTION_SUMMARY            	containing a modified Linsmaier and Skoog medium23,24 with Gamborg’s vitamins
CO:COLLECTION_SUMMARY            	(Sigma) and 5 mM MES buffer adjusted to pH 5.8. Fifty mM [U-13C6]glucose was
CO:COLLECTION_SUMMARY            	used as labeled substrate, and the composition of the remaining carbon and
CO:COLLECTION_SUMMARY            	nitrogen sources represented maternal phloem composition to minimize metabolic
CO:COLLECTION_SUMMARY            	perturbation and to maintain pseudo in vivo conditions as previously
CO:COLLECTION_SUMMARY            	described25. Silique culturing was performed in a 96-well plate with 0.3 mL of
CO:COLLECTION_SUMMARY            	medium and a single silique per well, under continuous light (250 µmol m-2
CO:COLLECTION_SUMMARY            	s-1). Tissue was collected and flash frozen immediately after each time point
CO:COLLECTION_SUMMARY            	(2, 4, 8, 16 and 32h). Uncultured siliques excised from the maternal plant were
CO:COLLECTION_SUMMARY            	used as unlabeled (0h) controls. Frozen tissue was sectioned, on top of dry ice,
CO:COLLECTION_SUMMARY            	to excise embryo from the siliques and to separate cotyledons from the embryo
CO:COLLECTION_SUMMARY            	axis. Cotyledon samples were extracted and analyzed for lipids in triplicates.
CO:COLLECTION_PROTOCOL_FILENAME  	13CLipids_CamelinaSeeds_Methods.docx
CO:SAMPLE_TYPE                   	Seeds
CO:COLLECTION_LOCATION           	Donald Danforth Plant Science Center
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Plants were grown in greenhouses with day/night temperature maintained at
TR:TREATMENT_SUMMARY             	22/20°C, 40-50% relative humidity, and 16h day/8h night photoperiod. Intact
TR:TREATMENT_SUMMARY             	siliques during the seed filling growth stage (15 days after fertilization) were
TR:TREATMENT_SUMMARY             	excised and placed in sterile media containing a modified Linsmaier and Skoog
TR:TREATMENT_SUMMARY             	medium23,24 with Gamborg’s vitamins (Sigma) and 5 mM MES buffer adjusted to pH
TR:TREATMENT_SUMMARY             	5.8. Fifty mM [U-13C6]glucose was used as labeled substrate, and the composition
TR:TREATMENT_SUMMARY             	of the remaining carbon and nitrogen sources represented maternal phloem
TR:TREATMENT_SUMMARY             	composition to minimize metabolic perturbation and to maintain pseudo in vivo
TR:TREATMENT_SUMMARY             	conditions as previously described25. Silique culturing was performed in a
TR:TREATMENT_SUMMARY             	96-well plate with 0.3 mL of medium and a single silique per well, under
TR:TREATMENT_SUMMARY             	continuous light (250 µmol m-2 s-1). Tissue was collected and flash frozen
TR:TREATMENT_SUMMARY             	immediately after each time point (2, 4, 8, 16 and 32h). Uncultured siliques
TR:TREATMENT_SUMMARY             	excised from the maternal plant were used as unlabeled (0h) controls. Frozen
TR:TREATMENT_SUMMARY             	tissue was sectioned, on top of dry ice, to excise embryo from the siliques and
TR:TREATMENT_SUMMARY             	to separate cotyledons from the embryo axis. Cotyledon samples were extracted
TR:TREATMENT_SUMMARY             	and analyzed for lipids in triplicates.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Frozen cotyledon samples from Camelina were homogenized using a tissue lyser and
SP:SAMPLEPREP_SUMMARY            	the extraction of lipids was carried out using a phase separation method
SP:SAMPLEPREP_SUMMARY            	previously described26. Briefly, 1 mL 7:3 methanol:chloroform (-20°C)
SP:SAMPLEPREP_SUMMARY            	containing the ultimateSPLASHTM ONE lipid mix (Avanti Polar lipids, Alabaster,
SP:SAMPLEPREP_SUMMARY            	AL) as internal standard (1:20 dilution) was added to the samples, vortexed
SP:SAMPLEPREP_SUMMARY            	vigorously and incubated on a rotary shaker for 2 hours at 4°C. After
SP:SAMPLEPREP_SUMMARY            	incubation, 500 µL of ice-cold water was added to the samples, vortexed and
SP:SAMPLEPREP_SUMMARY            	centrifuged at 14,000 rpm at 4°C for 10 min to achieve phase separation. The
SP:SAMPLEPREP_SUMMARY            	upper aqueous phase was carefully removed, 200 µL of methanol was added to the
SP:SAMPLEPREP_SUMMARY            	remaining organic phase containing lipids and centrifuged at 14,000 rpm for 5
SP:SAMPLEPREP_SUMMARY            	min to pellet the debris. The organic phase (supernatant) was transferred to a
SP:SAMPLEPREP_SUMMARY            	glass tube and dried using a speedvac centrifuge. Samples were re-suspended in
SP:SAMPLEPREP_SUMMARY            	200 µL of 49:49:2 acetonitrile: methanol: chloroform, filtered using 0.2 µm
SP:SAMPLEPREP_SUMMARY            	PTFE microcentrifuge filters and transferred to a glass vial for RPLC-HRMS
SP:SAMPLEPREP_SUMMARY            	analysis.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACTION_METHOD             	methanol:chloroform:water
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Separations for lipidomics were achieved using the loading pump of a Dionex
CH:CHROMATOGRAPHY_SUMMARY        	UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) operating at a flow
CH:CHROMATOGRAPHY_SUMMARY        	rate of 40 µL min-1 equipped with a custom-made C8 column (100 x 0.5 x 5 µm)
CH:CHROMATOGRAPHY_SUMMARY        	from Higgins Analytical Inc. (Mountain view, CA) re-packed from a nucleodur C8
CH:CHROMATOGRAPHY_SUMMARY        	Gravity column (Macherey-Nagel, Allentown, PA). Mobile phases comprised of 1% 1
CH:CHROMATOGRAPHY_SUMMARY        	M ammonium acetate, 0.1 % acetic acid in water (A) and 1% 1 M ammonium acetate,
CH:CHROMATOGRAPHY_SUMMARY        	0.1% acetic acid in 7:3 (v/v) acetonitrile: isopropanol (B). The following
CH:CHROMATOGRAPHY_SUMMARY        	gradient modified from a previously described method27 to adapt to micro flow
CH:CHROMATOGRAPHY_SUMMARY        	was used; 0-1 min at 55% B, 4 min at 75% B, 12 min at 89% B, 15 min at 99% B, 18
CH:CHROMATOGRAPHY_SUMMARY        	min at 99% B and 20 min at 55% B followed by equilibration up to 30 min.
CH:INSTRUMENT_NAME               	Dionex UltiMate 3000 RSLCnano
CH:COLUMN_NAME                   	Custom C8 - Higgins Analytical
CH:COLUMN_TEMPERATURE            	40
CH:FLOW_RATE                     	0.04 mL min-1
CH:INTERNAL_STANDARD             	Equisplash
CH:SOLVENT_A                     	1% 1 M ammonium acetate, 0.1 % acetic acid in water
CH:SOLVENT_B                     	1% 1 M ammonium acetate, 0.1% acetic acid in 7:3 (v/v) acetonitrile: isopropanol
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Fusion Tribrid Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	The eluent was sprayed on to the HESI source of an Orbitrap Fusion Lumos Tribrid
MS:MS_COMMENTS                   	MS, operated with sheath gas, 25 arbitrary units; auxiliary gas, 5 arbitrary
MS:MS_COMMENTS                   	units; ion transfer tube temperature, 300oC; vaporizer temperature, 100oC; and
MS:MS_COMMENTS                   	S-lens RF level, 60. The spray voltage was 4 kV in both positive and negative
MS:MS_COMMENTS                   	modes. Full MS data were collected for mass ranges 450-1200 m/z at 240,000
MS:MS_COMMENTS                   	resolution from both positive and negative modes simultaneously, using polarity
MS:MS_COMMENTS                   	switch. The AGC target was set to “Standard” and the maximum IT was set to
MS:MS_COMMENTS                   	100 ms.
MS:ION_MODE                      	NEGATIVE
MS:MS_RESULTS_FILE               	ST002239_AN003656_Results.txt	UNITS:Intensity	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END