{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002992","ANALYSIS_ID":"AN004912","VERSION":"1","CREATED_ON":"October 2, 2023, 5:07 pm"},

"PROJECT":{"PROJECT_TITLE":"Folate depletion induces erythroid differentiation through perturbation of de novo purine synthesis","PROJECT_SUMMARY":"Folate, an essential vitamin, is a one-carbon acceptor and donor in key metabolic reactions. Erythroid cells harbor a unique sensitivity to folate deprivation, as revealed by the primary pathological manifestation of nutritional folate deprivation: megaloblastic anemia. To study this metabolic sensitivity, we applied mild folate depletion to human and mouse erythroid cell lines, and primary murine erythroid progenitors. We show that folate depletion induces early blockade of purine synthesis and accumulation of the purine synthesis intermediate and signaling molecule, AICAR, followed by enhanced heme metabolism, hemoglobin synthesis, and erythroid differentiation. This is phenocopied by inhibition of folate metabolism using the SHMT1/2 inhibitor - SHIN1, and by AICAR supplementation. Mechanistically, the metabolically-driven differentiation is independent of nucleotide sensing through mTORC1 and AMPK, and is instead mediated by protein kinase C (PKC). Our findings suggest that folate deprivation-induced premature differentiation of erythroid progenitor cells is a molecular etiology to folate-deficiency induced anemia.","INSTITUTE":"Boston Children''s Hospital, Harvard Medical School","DEPARTMENT":"pathology","LABORATORY":"Kanarek Lab","LAST_NAME":"Kanarek","FIRST_NAME":"Naama","ADDRESS":"Enders 1116.2, 300 Longwood Ave, Boston, MA 02115","EMAIL":"naama.kanarek@childrens.harvard.edu","PHONE":"(617) 355-7433"},

"STUDY":{"STUDY_TITLE":"Polar metabolite levels in K562 cells following C13-Serine and C13-Glycine tracing","STUDY_SUMMARY":"Culture of K562 cells for 7 days in RPMI media containing 2000 nM folic acid or 100 nM folic acid. At day 8, media was changed to 2000 nM or 100 nM folic acid with unlabeled serine and glycine, or 2-C13-Serine and 2-C13-Glycine at RPMI levels. Amino acid tracing was performed for 24 hours. These samples were followed up by metabolomics targeting polar metabolites.","INSTITUTE":"Boston Children''s Hospital, Harvard Medical School","DEPARTMENT":"pathology","LABORATORY":"Kanarek Lab","LAST_NAME":"Kanarek","FIRST_NAME":"Naama","ADDRESS":"Enders 1116.2, 300 Longwood Ave, Boston, MA 02115","EMAIL":"naama.kanarek@childrens.harvard.edu","PHONE":"6173557433","NUM_GROUPS":"6"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_STRAIN_DETAILS":"K-562"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_Unlabeled_HILIC_1",
"Factors":{"Treatment":"2000nM_FA_Unlabeled"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM447.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_Unlabeled_HILIC_2",
"Factors":{"Treatment":"2000nM_FA_Unlabeled"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM448.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_Unlabeled_HILIC_3",
"Factors":{"Treatment":"2000nM_FA_Unlabeled"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM449.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_Unlabeled_HILIC_4",
"Factors":{"Treatment":"2000nM_FA_Unlabeled"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM450.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_Unlabeled_HILIC_1",
"Factors":{"Treatment":"100nM_FA_Unlabeled"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM451.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_Unlabeled_HILIC_2",
"Factors":{"Treatment":"100nM_FA_Unlabeled"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM452.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_Unlabeled_HILIC_3",
"Factors":{"Treatment":"100nM_FA_Unlabeled"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM453.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_Unlabeled_HILIC_4",
"Factors":{"Treatment":"100nM_FA_Unlabeled"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM454.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_C13_Glycine_HILIC_1",
"Factors":{"Treatment":"2000nM_FA_C13_Glycine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM455.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_C13_Glycine_HILIC_2",
"Factors":{"Treatment":"2000nM_FA_C13_Glycine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM456.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_C13_Glycine_HILIC_3",
"Factors":{"Treatment":"2000nM_FA_C13_Glycine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM457.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_C13_Glycine_HILIC_4",
"Factors":{"Treatment":"2000nM_FA_C13_Glycine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM458.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_C13_Serine_HILIC_1",
"Factors":{"Treatment":"2000nM_FA_C13_Serine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM459.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_C13_Serine_HILIC_2",
"Factors":{"Treatment":"2000nM_FA_C13_Serine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM460.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_C13_Serine_HILIC_3",
"Factors":{"Treatment":"2000nM_FA_C13_Serine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM461.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_FA_C13_Serine_HILIC_4",
"Factors":{"Treatment":"2000nM_FA_C13_Serine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM462.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_C13_Glycine_HILIC_1",
"Factors":{"Treatment":"100nM_FA_C13_Glycine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM467.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_C13_Glycine_HILIC_2",
"Factors":{"Treatment":"100nM_FA_C13_Glycine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM468.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_C13_Glycine_HILIC_3",
"Factors":{"Treatment":"100nM_FA_C13_Glycine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM469.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_C13_Glycine_HILIC_4",
"Factors":{"Treatment":"100nM_FA_C13_Glycine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM470.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_C13_Serine_HILIC_1",
"Factors":{"Treatment":"100nM_FA_C13_Serine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM471.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_C13_Serine_HILIC_2",
"Factors":{"Treatment":"100nM_FA_C13_Serine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM472.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_C13_Serine_HILIC_3",
"Factors":{"Treatment":"100nM_FA_C13_Serine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM473.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_FA_C13_Serine_HILIC_4",
"Factors":{"Treatment":"100nM_FA_C13_Serine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210903_QEF1_HILIC_SGtracing_AM474.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"One million cells from culture were collected via centrifugation for 20 seconds at 18,000xG, washed with 0.9% NaCl, and collected via centrifugation for 20 seconds at 18,000xG","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Culture of K562 cells for 7 days in RPMI media containing 2000 nM folic acid or 100 nM folic acid. At day 8, media was changed to 2000 nM or 100 nM folic acid with unlabeled serine and glycine, or 2-C13-Serine and 2-C13-Glycine at RPMI levels. Amino acid tracing was performed for 24 hours. The level of serine and glycine in all conditions was 30 and 10 mg/L, respectively."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Pellet was resuspended in extraction buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000 g to pellet cell debris. The supernatant was divided into two tubes and dried on ice using a liquid nitrogen dryer. One tube of dried sample was used for polar metabolite detection. Dried samples were resuspended in 25 μL water"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Sample was injected into a ZIC-pHILIC 150 x 2.1 mm (5 μm particle size) column (EMD Millipore). operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separation was achieved using the following conditions: buffer A was acetonitrile; buffer B was 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water; resulting pH is around 9 without pH adjustment. Gradient conditions we used were: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"EMD Millipore ZIC-HILIC (100 x 2.1 mm,3.5 um)","SOLVENT_A":"100% acetonitrile","SOLVENT_B":"100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide","FLOW_GRADIENT":"linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate","FLOW_RATE":"150 mL/min","COLUMN_TEMPERATURE":"25"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"Polar metabolites were relatively quantified while referencing an in-house library of chemical standards and using TraceFinder 4.1 (Thermo Fisher Scientific, Waltham, MA, USA), with a 5 ppm mass tolerance."},

"MS_METABOLITE_DATA":{
"Units":"peak area",

"Data":[{"Metabolite":"glycine_C0","K562_2000nM_FA_Unlabeled_HILIC_1":"28910945.26","K562_2000nM_FA_Unlabeled_HILIC_2":"28323568.93","K562_2000nM_FA_Unlabeled_HILIC_3":"27571245.91","K562_2000nM_FA_Unlabeled_HILIC_4":"27065018.43","K562_100nM_FA_Unlabeled_HILIC_1":"25190836","K562_100nM_FA_Unlabeled_HILIC_2":"27014723.83","K562_100nM_FA_Unlabeled_HILIC_3":"23604673.07","K562_100nM_FA_Unlabeled_HILIC_4":"22953418.25","K562_2000nM_FA_C13_Glycine_HILIC_1":"21816207.58","K562_2000nM_FA_C13_Glycine_HILIC_2":"19008551.77","K562_2000nM_FA_C13_Glycine_HILIC_3":"22771368.28","K562_2000nM_FA_C13_Glycine_HILIC_4":"20427261.9","K562_2000nM_FA_C13_Serine_HILIC_1":"15088200.61","K562_2000nM_FA_C13_Serine_HILIC_2":"17822057.66","K562_2000nM_FA_C13_Serine_HILIC_3":"15469104.78","K562_2000nM_FA_C13_Serine_HILIC_4":"16345976.16","K562_100nM_FA_C13_Glycine_HILIC_1":"16786035.4","K562_100nM_FA_C13_Glycine_HILIC_2":"18731987.73","K562_100nM_FA_C13_Glycine_HILIC_3":"20484264.62","K562_100nM_FA_C13_Glycine_HILIC_4":"15772562.88","K562_100nM_FA_C13_Serine_HILIC_1":"15880532.59","K562_100nM_FA_C13_Serine_HILIC_2":"14576885.27","K562_100nM_FA_C13_Serine_HILIC_3":"15152088.72","K562_100nM_FA_C13_Serine_HILIC_4":"14812292.91"},{"Metabolite":"glycine_C1","K562_2000nM_FA_Unlabeled_HILIC_1":"195658.7077","K562_2000nM_FA_Unlabeled_HILIC_2":"138845.2247","K562_2000nM_FA_Unlabeled_HILIC_3":"115086.6486","K562_2000nM_FA_Unlabeled_HILIC_4":"680209.7138","K562_100nM_FA_Unlabeled_HILIC_1":"304030.6308","K562_100nM_FA_Unlabeled_HILIC_2":"602041.4227","K562_100nM_FA_Unlabeled_HILIC_3":"449411.3482","K562_100nM_FA_Unlabeled_HILIC_4":"380517.2598","K562_2000nM_FA_C13_Glycine_HILIC_1":"7218146.3","K562_2000nM_FA_C13_Glycine_HILIC_2":"6766689.128","K562_2000nM_FA_C13_Glycine_HILIC_3":"6784921.782","K562_2000nM_FA_C13_Glycine_HILIC_4":"6403520.731","K562_2000nM_FA_C13_Serine_HILIC_1":"15791003.97","K562_2000nM_FA_C13_Serine_HILIC_2":"17063561.49","K562_2000nM_FA_C13_Serine_HILIC_3":"16985904.84","K562_2000nM_FA_C13_Serine_HILIC_4":"17620964.99","K562_100nM_FA_C13_Glycine_HILIC_1":"8020342.687","K562_100nM_FA_C13_Glycine_HILIC_2":"8625825.808","K562_100nM_FA_C13_Glycine_HILIC_3":"9616263.459","K562_100nM_FA_C13_Glycine_HILIC_4":"4511892.886","K562_100nM_FA_C13_Serine_HILIC_1":"10667473.56","K562_100nM_FA_C13_Serine_HILIC_2":"9611142.869","K562_100nM_FA_C13_Serine_HILIC_3":"10040128.8","K562_100nM_FA_C13_Serine_HILIC_4":"9376719.967"},{"Metabolite":"serine_C0","K562_2000nM_FA_Unlabeled_HILIC_1":"23728906.68","K562_2000nM_FA_Unlabeled_HILIC_2":"20714183.64","K562_2000nM_FA_Unlabeled_HILIC_3":"18761904.75","K562_2000nM_FA_Unlabeled_HILIC_4":"20210119.21","K562_100nM_FA_Unlabeled_HILIC_1":"43001237.82","K562_100nM_FA_Unlabeled_HILIC_2":"44647401.71","K562_100nM_FA_Unlabeled_HILIC_3":"41002145.67","K562_100nM_FA_Unlabeled_HILIC_4":"42874227.12","K562_2000nM_FA_C13_Glycine_HILIC_1":"19436809.2","K562_2000nM_FA_C13_Glycine_HILIC_2":"17517434.16","K562_2000nM_FA_C13_Glycine_HILIC_3":"20157826.69","K562_2000nM_FA_C13_Glycine_HILIC_4":"17996333.57","K562_2000nM_FA_C13_Serine_HILIC_1":"12531166.75","K562_2000nM_FA_C13_Serine_HILIC_2":"12725181.23","K562_2000nM_FA_C13_Serine_HILIC_3":"13228048.54","K562_2000nM_FA_C13_Serine_HILIC_4":"16925803.27","K562_100nM_FA_C13_Glycine_HILIC_1":"38952767.76","K562_100nM_FA_C13_Glycine_HILIC_2":"40460591.25","K562_100nM_FA_C13_Glycine_HILIC_3":"45012678.05","K562_100nM_FA_C13_Glycine_HILIC_4":"75282338.96","K562_100nM_FA_C13_Serine_HILIC_1":"23240876.77","K562_100nM_FA_C13_Serine_HILIC_2":"21887183.99","K562_100nM_FA_C13_Serine_HILIC_3":"22570229.68","K562_100nM_FA_C13_Serine_HILIC_4":"22988202.26"},{"Metabolite":"serine_C1","K562_2000nM_FA_Unlabeled_HILIC_1":"426134.5593","K562_2000nM_FA_Unlabeled_HILIC_2":"320826.1607","K562_2000nM_FA_Unlabeled_HILIC_3":"292344.8723","K562_2000nM_FA_Unlabeled_HILIC_4":"288268.4807","K562_100nM_FA_Unlabeled_HILIC_1":"1085644.903","K562_100nM_FA_Unlabeled_HILIC_2":"1322206.413","K562_100nM_FA_Unlabeled_HILIC_3":"845890.738","K562_100nM_FA_Unlabeled_HILIC_4":"1290215.339","K562_2000nM_FA_C13_Glycine_HILIC_1":"1969774.641","K562_2000nM_FA_C13_Glycine_HILIC_2":"2002676.178","K562_2000nM_FA_C13_Glycine_HILIC_3":"1505230.784","K562_2000nM_FA_C13_Glycine_HILIC_4":"1722314.939","K562_2000nM_FA_C13_Serine_HILIC_1":"7035208.931","K562_2000nM_FA_C13_Serine_HILIC_2":"7593615.542","K562_2000nM_FA_C13_Serine_HILIC_3":"6980693.515","K562_2000nM_FA_C13_Serine_HILIC_4":"7449906.862","K562_100nM_FA_C13_Glycine_HILIC_1":"3668391.027","K562_100nM_FA_C13_Glycine_HILIC_2":"3991050.416","K562_100nM_FA_C13_Glycine_HILIC_3":"4268807.998","K562_100nM_FA_C13_Glycine_HILIC_4":"3727105.622","K562_100nM_FA_C13_Serine_HILIC_1":"21331787.85","K562_100nM_FA_C13_Serine_HILIC_2":"19522694.32","K562_100nM_FA_C13_Serine_HILIC_3":"19712834.15","K562_100nM_FA_C13_Serine_HILIC_4":"19260639.09"},{"Metabolite":"IMP_C0","K562_2000nM_FA_Unlabeled_HILIC_1":"1624064.619","K562_2000nM_FA_Unlabeled_HILIC_2":"1540143.995","K562_2000nM_FA_Unl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"Metabolites":[{"metabolite_name":"glycine_C0"},{"metabolite_name":"glycine_C1"},{"metabolite_name":"serine_C0"},{"metabolite_name":"serine_C1"},{"metabolite_name":"IMP_C0"},{"metabolite_name":"IMP_C1"},{"metabolite_name":"GSSG_C0"},{"metabolite_name":"GSSG_C1"},{"metabolite_name":"GSSG_C2"},{"metabolite_name":"GSH_Ellmans_C0"},{"metabolite_name":"GSH_Ellmans_C1"}]
}

}