{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002270","ANALYSIS_ID":"AN003711","VERSION":"1","CREATED_ON":"02-08-2024"},

"PROJECT":{"PROJECT_TITLE":"Glucose metabolism during Xenopus Regeneration","PROJECT_SUMMARY":"Targeted metabolomics of Xenopus tropicalis to study glucose metabolism during regeneration","INSTITUTE":"University of Washington","DEPARTMENT":"Biochemistry","LABORATORY":"Wills Lab","LAST_NAME":"Patel","FIRST_NAME":"Jeet","ADDRESS":"1705 NE Pacific St., HSB J-Wing, J405, Seattle, Washington, 98195, USA","EMAIL":"pateljeet1224@gmail.com","PHONE":"2065431748","FUNDING_SOURCE":"Royalty Research Fund, 1R01NS099124 from NINDS","DOI":"http://dx.doi.org/10.21228/M81Q5B"},

"STUDY":{"STUDY_TITLE":"Xenopus tropicalis glycolysis and PPP inhibition","STUDY_SUMMARY":"Stage 41 tadpoles were injected with 4 nmol of the glycolysis inhibitor, 2-deoxyglucose (2DG), or a tracer control to evaluate consequences of inhibition on metabolites 24 hours after treatment began. Similarly, tadpoles were incubated in DMSO control or 1 of 2 G6PD inhibitors (Dehydroepiandrosterone and g6pdi), to similarly assess the consequences of inhibiting the pentose phosphate pathway. We find that inhibition of glucose metabolism with 2DG results in a decrease in downstream glycolytic intermediates, confirming a reduction in activity of this pathway. G6PD inhibition was not as clear as changes were less consistent across treatments and downstream metabolites did not behave in a coordinated way, though impacts on other metabolic processes by these inhibitors may be fruitful for exploring how they perturb metabolism in the tadpoles.","INSTITUTE":"University of Washington","LAST_NAME":"Patel","FIRST_NAME":"Jeet","ADDRESS":"1705 NE Pacific St., HSB J-Wing, J405, Seattle, Washington, 98195, USA","EMAIL":"pateljeet1224@gmail.com","PHONE":"2065431748","SUBMIT_DATE":"2022-08-03"},

"SUBJECT":{"SUBJECT_TYPE":"Other organism","SUBJECT_SPECIES":"Xenopus tropicalis","AGE_OR_AGE_RANGE":"Stage 41-43"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"11",
"Sample ID":"2DG1",
"Factors":{"Condition":"2DG"},
"Additional sample data":{"Replicate":"1","RAW_FILE_NAME":"2022-05-09_Wills-20-11_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-11_NEG.mzML"}
},
{
"Subject ID":"16",
"Sample ID":"2DG2",
"Factors":{"Condition":"2DG"},
"Additional sample data":{"Replicate":"2","RAW_FILE_NAME":"2022-05-09_Wills-20-16_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-16_NEG.mzML"}
},
{
"Subject ID":"10",
"Sample ID":"2DG3",
"Factors":{"Condition":"2DG"},
"Additional sample data":{"Replicate":"3","RAW_FILE_NAME":"2022-05-09_Wills-20-10_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-10_NEG.mzML"}
},
{
"Subject ID":"17",
"Sample ID":"2DG4",
"Factors":{"Condition":"2DG"},
"Additional sample data":{"Replicate":"4","RAW_FILE_NAME":"2022-05-09_Wills-20-17_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-17_NEG.mzML"}
},
{
"Subject ID":"12",
"Sample ID":"DHEA1",
"Factors":{"Condition":"DHEA"},
"Additional sample data":{"Replicate":"1","RAW_FILE_NAME":"2022-05-09_Wills-20-12_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-12_NEG.mzML"}
},
{
"Subject ID":"1",
"Sample ID":"DHEA2",
"Factors":{"Condition":"DHEA"},
"Additional sample data":{"Replicate":"2","RAW_FILE_NAME":"2022-05-09_Wills-20-1_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-1_NEG.mzML"}
},
{
"Subject ID":"5",
"Sample ID":"DHEA3",
"Factors":{"Condition":"DHEA"},
"Additional sample data":{"Replicate":"3","RAW_FILE_NAME":"2022-05-09_Wills-20-5_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-5_NEG.mzML"}
},
{
"Subject ID":"18",
"Sample ID":"DHEA4",
"Factors":{"Condition":"DHEA"},
"Additional sample data":{"Replicate":"4","RAW_FILE_NAME":"2022-05-09_Wills-20-18_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-18_NEG.mzML"}
},
{
"Subject ID":"8",
"Sample ID":"DMSO1",
"Factors":{"Condition":"DMSO"},
"Additional sample data":{"Replicate":"1","RAW_FILE_NAME":"2022-05-09_Wills-20-8_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-8_NEG.mzML"}
},
{
"Subject ID":"20",
"Sample ID":"DMSO2",
"Factors":{"Condition":"DMSO"},
"Additional sample data":{"Replicate":"2","RAW_FILE_NAME":"2022-05-09_Wills-20-20_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-20_NEG.mzML"}
},
{
"Subject ID":"13",
"Sample ID":"DMSO3",
"Factors":{"Condition":"DMSO"},
"Additional sample data":{"Replicate":"3","RAW_FILE_NAME":"2022-05-09_Wills-20-13_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-13_NEG.mzML"}
},
{
"Subject ID":"6",
"Sample ID":"DMSO4",
"Factors":{"Condition":"DMSO"},
"Additional sample data":{"Replicate":"4","RAW_FILE_NAME":"2022-05-09_Wills-20-6_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-6_NEG.mzML"}
},
{
"Subject ID":"7",
"Sample ID":"g6pdi1",
"Factors":{"Condition":"g6pdi"},
"Additional sample data":{"Replicate":"1","RAW_FILE_NAME":"2022-05-09_Wills-20-7_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-7_NEG.mzML"}
},
{
"Subject ID":"3",
"Sample ID":"g6pdi2",
"Factors":{"Condition":"g6pdi"},
"Additional sample data":{"Replicate":"2","RAW_FILE_NAME":"2022-05-09_Wills-20-3_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-3_NEG.mzML"}
},
{
"Subject ID":"15",
"Sample ID":"g6pdi3",
"Factors":{"Condition":"g6pdi"},
"Additional sample data":{"Replicate":"3","RAW_FILE_NAME":"2022-05-09_Wills-20-15_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-15_NEG.mzML"}
},
{
"Subject ID":"9",
"Sample ID":"g6pdi4",
"Factors":{"Condition":"g6pdi"},
"Additional sample data":{"Replicate":"4","RAW_FILE_NAME":"2022-05-09_Wills-20-9_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-9_NEG.mzML"}
},
{
"Subject ID":"14",
"Sample ID":"MR1",
"Factors":{"Condition":"MR"},
"Additional sample data":{"Replicate":"1","RAW_FILE_NAME":"2022-05-09_Wills-20-14_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-14_NEG.mzML"}
},
{
"Subject ID":"2",
"Sample ID":"MR2",
"Factors":{"Condition":"MR"},
"Additional sample data":{"Replicate":"2","RAW_FILE_NAME":"2022-05-09_Wills-20-2_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-2_NEG.mzML"}
},
{
"Subject ID":"4",
"Sample ID":"MR3",
"Factors":{"Condition":"MR"},
"Additional sample data":{"Replicate":"3","RAW_FILE_NAME":"2022-05-09_Wills-20-4_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-4_NEG.mzML"}
},
{
"Subject ID":"19",
"Sample ID":"MR4",
"Factors":{"Condition":"MR"},
"Additional sample data":{"Replicate":"4","RAW_FILE_NAME":"2022-05-09_Wills-20-19_POS.mzML","RAW_FILE_NAME":"2022-05-09_Wills-20-19_NEG.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Tadpoles were anesthetized with 0.05% MS-222 in 1/9x MR and tested for response to touch prior to tissue collection. Injections of miniRuby or 4nmol of 2DG were performed or tadpoles were incubated in media with DMSO, DHEA, or g6pdi/ 10 whole animals were collected at 24 hours post treatment, media was removed, and samples were frozen on dry ice immediately.","SAMPLE_TYPE":"Stage 43 tadpoles"},

"TREATMENT":{"TREATMENT_SUMMARY":"Dihydroepiandrosterone (BioVision, 2172) was resuspended to a 1M stock in DMSO and g6pdi (Cayman Chemical Co., 31484) to a 10mM stock in DMSO. Tadples were reared with 0.1% DMSO, 25µM DHEA or 10µM g6pdi diluted in 1/9x MR until collection after a 24 treatment. For 2DG injections, wild type tadpoles at stage 41 were anesthetized with MS-222 and moved from culture dish to a thickly coasted agarose dish in one drop of media. Excess media was removed. Using a microinjector, a pulled needle containing vMO and labeled dextran tracer was inserted into the ventral tail vein and 2×2 nL of 1M 2DG (Sigma, D8375) were injected. Controls for 2DG experiments were injected with equal volumes of tracer. Embryos were returned to fresh media and screened for tracer fluorescence in the bloodstream. Injected animals were kept in 1/9x MR until collection after a 24 treatment."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Aqueous metabolites for targeted LC-MS analysis were extracted using a protein precipitation method similar to the one described elsewhere (Mathon et al., 2019; Meador et al., 2020). Samples were first homogenized in 200 µL purified deionized water at 4 ˚C, and then 800 µL of methanol containing 6C13-glucose and 2C13-glutamate (reference internal standards) was added. Afterwards samples were vortexed, stored for 30 minutes at -20 ˚C, sonicated in an ice bath for 10 minutes, centrifuged for 15 min at 14,000 rpm and 4 ˚C, and then 600 µL of supernatant was collected from each sample. Lastly, recovered supernatants were dried on a SpeedVac and reconstituted in 1.0 mL of LC-matching solvent containing 2C13-tyrosine and 3C13-lactate (reference internal standards).","SAMPLEPREP_PROTOCOL_FILENAME":"JPatel_LC-MS_Methods.pdf"},

"CHROMATOGRAPHY":{"INSTRUMENT_NAME":"Shimadzu Nexera X2","COLUMN_NAME":"Waters XBridge BEH Amide XP","CHROMATOGRAPHY_TYPE":"HILIC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","ANALYSIS_PROTOCOL_FILE":"JPatel_LC-MS_Methods.pdf"},

"MS":{"INSTRUMENT_NAME":"ABI Sciex 6500+","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","MS_COMMENTS":"MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. The LC-MS assay was targeting 361 metabolites (plus 4 spiked reference internal standards).","ION_MODE":"NEGATIVE"},

"MS_METABOLITE_DATA":{
"Units":"Peak Intensity",

"Data":[{"Metabolite":"1C13-Citrulline","2DG1":"","2DG2":"","2DG3":"","2DG4":"","DHEA1":"","DHEA2":"","DHEA3":"","DHEA4":"","DMSO1":"","DMSO2":"","DMSO3":"","g6pdi1":"","g6pdi2":"","g6pdi3":"","g6pdi4":"","MR1":"","MR2":"","MR3":"","MR4":""},{"Metabolite":"2_3-Dihydroxybenzoic Acid","2DG1":"","2DG2":"","2DG3":"","2DG4":"","DHEA1":"","DHEA2":"","DHEA3":"","DHEA4":"","DMSO1":"","DMSO2":"","DMSO3":"","g6pdi1":"","g6pdi2":"","g6pdi3":"","g6pdi4":"","MR1":"","MR2":"","MR3":"","MR4":""},{"Metabolite":"2/3-Phosphoglyceric 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