{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003023","ANALYSIS_ID":"AN004957","VERSION":"1","CREATED_ON":"December 19, 2023, 8:12 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":"Porphyrin metabolite levels in K562 cells following 6 day culture in 2,000 nM or 100 nM folic acid","STUDY_SUMMARY":"Culture of K562 cells in RPMI media containing 2000 nM or 100 nM folic acid for 6 days. These samples were followed up by metabolomics targeting porphyrin 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":"5"},

"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_Porphyrin_1",
"Factors":{"Folic_Acid_Concentration":"2000_nM"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM278.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_Porphyrin_2",
"Factors":{"Folic_Acid_Concentration":"2000_nM"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM279.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_Porphyrin_3",
"Factors":{"Folic_Acid_Concentration":"2000_nM"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM280.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_2000nM_Porphyrin_4",
"Factors":{"Folic_Acid_Concentration":"2000_nM"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM281.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_Porpyrin_1",
"Factors":{"Folic_Acid_Concentration":"100_nM"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM282.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_Porpyrin_2",
"Factors":{"Folic_Acid_Concentration":"100_nM"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM283.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_Porpyrin_3",
"Factors":{"Folic_Acid_Concentration":"100_nM"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM284.raw"}
},
{
"Subject ID":"K562",
"Sample ID":"K562_100nM_Porpyrin_4",
"Factors":{"Folic_Acid_Concentration":"100_nM"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM285.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 6 days in RPMI media containing 2000 nM or 100 nM folic acid for 6 days. These samples were followed up by metabolomics targeting porphyrin metabolites."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"One million cells from culture were collected via centrifugation, washed with 0.9% NaCl, and resuspended in 150 µl of porphyrin extraction buffer (1:4 ratio of 1.7 M HCl:ACN, 1µM deuteroporphyrin IX (Frontier Scientific, D510-9)) and 0.5 µM isotopically labeled amino acids (Cambridge Isotopes, MSK-A2-1.2)). Samples were vigorously shaken for 20 min at 16ºC in a thermomixer (Eppendorf), sonicated for 10 cycles at 4ºC with 30 sec on and 30 sec off, then incubated at 4ºC for 10 min. Following incubation on ice, samples were centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was collected and 40.5 µl super-saturated MgSO4 and 12 µl 5 M NaCl were added. Samples were vortexed for 30 sec and further shaken for 10 min at 16 ºC in a thermomixer. Finally, a 10 min 10,000 rpm centrifugation was used to separate the organic layer (upper) from the aqueous layer (lower). The upper organic layer was collected."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Sample (5 uL) was injected onto a 2.6 μm, 150 x 3 mm C18 column (Phenomenex, 00F-4462-Y0) equipped with a 3.0 mm safe-guard column (Phenomenex, AJ0-8775). Column compartment was heated to 45 ºC. Porphyrins were separated with a chromatographic gradient at a flow rate of 0.800 ml min−1 as follows: 0–2 min: 5% B; 2-19min: linear gradient from 5% to 95% B; 19-21min: 95% B; 21.1-23min: return to 5% B.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex C18 (150 x 3mm,2.6um)","SOLVENT_A":"95% water/5% acetonitrile; 0.1% formic acid","SOLVENT_B":"5% water/95% acetonitrile; 0.1% formic acid","FLOW_GRADIENT":"linear gradient from 5% to 95%","FLOW_RATE":"0.8 mL/min","COLUMN_TEMPERATURE":"45"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The mass spectrometer was operated in full-scan, positive ionization mode using a narrow-range scan: 450-700m/z, with an additional tSIM scan for hemin (616.1767 m/z), CoproP (655.2762 m/z), and PPIX (563.2653 m/z). 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":"Arbitrary Units",

"Data":[{"Metabolite":"Biliverdin","K562_2000nM_Porphyrin_1":"190999.5034","K562_2000nM_Porphyrin_2":"277219.3206","K562_2000nM_Porphyrin_3":"213790.6119","K562_2000nM_Porphyrin_4":"228208.118","K562_100nM_Porpyrin_1":"256222.7501","K562_100nM_Porpyrin_2":"280173.5827","K562_100nM_Porpyrin_3":"227497.6816","K562_100nM_Porpyrin_4":"254174.1875"},{"Metabolite":"Heme","K562_2000nM_Porphyrin_1":"15060580.3","K562_2000nM_Porphyrin_2":"12080916.52","K562_2000nM_Porphyrin_3":"15723822.73","K562_2000nM_Porphyrin_4":"13846413.2","K562_100nM_Porpyrin_1":"69215619.06","K562_100nM_Porpyrin_2":"76706810.94","K562_100nM_Porpyrin_3":"67696074.88","K562_100nM_Porpyrin_4":"74083933.98"},{"Metabolite":"Protoporphyrin IX","K562_2000nM_Porphyrin_1":"24415.38995","K562_2000nM_Porphyrin_2":"32860.48443","K562_2000nM_Porphyrin_3":"40204.07607","K562_2000nM_Porphyrin_4":"23893.14971","K562_100nM_Porpyrin_1":"116093.4279","K562_100nM_Porpyrin_2":"129099.3213","K562_100nM_Porpyrin_3":"98061.14129","K562_100nM_Porpyrin_4":"115579.2699"}],

"Metabolites":[{"Metabolite":"Biliverdin","Standardized name":"Biliverdin","Formula":"C33H34N4O6","Exact mass":"582.2478","Sub class":"Bilirubins"},{"Metabolite":"Heme","Standardized name":"Heme","Formula":"C34H32FeN4O4","Exact mass":"616.1773","Sub class":"Metalloporphyrins"},{"Metabolite":"Protoporphyrin IX","Standardized name":"Protoporphyrin","Formula":"C34H34N4O4","Exact mass":"562.2580","Sub class":"Porphyrins"}]
}

}