{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002900","ANALYSIS_ID":"AN004759","VERSION":"1","CREATED_ON":"October 1, 2023, 9:09 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":"Folate metabolite levels in erythroid progenitor cells following SHIN1 and inosine treatment","STUDY_SUMMARY":"Culture of erythroid progenitor cells cultured in SFEM II media supplemented with SHIN1 and/or inosine for 48 hours followed up by metabolomics targeting folate 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":"4"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","CELL_BIOSOURCE_OR_SUPPLIER":"Lineage depleted erythroid progenitor cells isolated from E14.5 mice."},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_Vehicle_folate_1",
"Factors":{"Treatment":"Vehicle"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM916.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_Vehicle_folate_2",
"Factors":{"Treatment":"Vehicle"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM917.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_Vehicle_folate_3",
"Factors":{"Treatment":"Vehicle"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM918.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_SHIN1_folate_1",
"Factors":{"Treatment":"SHIN1"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM919.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_SHIN1_folate_2",
"Factors":{"Treatment":"SHIN1"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM920.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_SHIN1_folate_3",
"Factors":{"Treatment":"SHIN1"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM921.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_Inosine_folate_1",
"Factors":{"Treatment":"Inosine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM922.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_Inosine_folate_2",
"Factors":{"Treatment":"Inosine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM923.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_Inosine_folate_3",
"Factors":{"Treatment":"Inosine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM924.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_SHIN1_Inosine_folate_1",
"Factors":{"Treatment":"SHIN1_Inosine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM925.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_SHIN1_Inosine_folate_2",
"Factors":{"Treatment":"SHIN1_Inosine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM926.raw"}
},
{
"Subject ID":"Erythroid_Progenitor",
"Sample ID":"EP_SHIN1_Inosine_folate_3",
"Factors":{"Treatment":"SHIN1_Inosine"},
"Additional sample data":{"RAW_FILE_NAME":"Copy of 20220715_AM_ErythProg3_Folates_AM927.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":"Erythroid progenitor cells were cultured for 48hr in SFEM II media containing vehicle, SHIN1 (1.25 uM), inosine (100 uM), or SHIN1+inosine."},

"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 folate metabolite detection. Folate samples were resuspended in reconstitution buffer (0.5% ascorbic acid, 1% K2HPO4 and 0.5% 2-mercaptoethanol) containing rat serum (Sigma, R9759). The rat serum contains the enzymes required to strip the polyglutamate tail from the measured folate. Rat endogenous folate was removed from the rat serum by activated carbon treatment (Sigma, C9157). Polyglutamate tail removal was carried out for 2 h at 37 °C. After the polyglutamate tail removal, the pH was adjusted to 4 using formic acid and samples were loaded on Bond Elute-pH columns (Agilent, 14102062) for a pH-based clean-up. Columns were eluted with 300 μl elution buffer (50% methanol, 0.25% NH4OAc, 0.5% 2-mercaptoethanol), followed by drying the samples using a liquid nitrogen dryer manifold and resuspension in 25 μl water."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"5 μl 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). The column oven and autosampler tray were held at 30 °C and 4 °C, respectively. The following conditions were used to achieve chromatographic separation: buffer A was 0.1% formic acid; buffer B was acetonitrile with 0.1% formic acid. The chromatographic gradient was run at a flow rate of 0.250 ml min−1 as follows: 0–5 min: gradient was held at 5% B; 5–10 min: linear gradient of 5% to 36% B; 10.1–14.0 min: linear gradient from 36–95% B; 14.1–18.0 min: gradient was returned to 5% B.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)","SOLVENT_A":"0.1% formic acid","SOLVENT_B":"acetonitrile with 0.1% formic acid","FLOW_GRADIENT":"0.250 ml min−1 as follows: 0–5 min: gradient was held at 5% B; 5–10 min: linear gradient of 5% to 36% B; 10.1–14.0 min: linear gradient from 36–95% B; 14.1–18.0 min: gradient was returned to 5% B.","FLOW_RATE":"250 uL/min","COLUMN_TEMPERATURE":"30"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"The mass spectrometer was operated in full-scan, positive ionization mode using three narrow-range scans: 438–450 m/z; 452–462 m/z; and 470–478 m/z, with the resolution set at 70,000, the AGC target at 10e6, and the maximum injection time of 150 ms. HESI settings were: sheath gas flow rate: 40; Aux gas flow rate: 10; Sweep gas: 0; Spray voltage: 2.8 (neg) 3.5 (pos); Capillary temperature 300; S-lens RF level 50; Aux gas heater temp: 350."},

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

"Data":[{"Metabolite":"5-methyltetrahydrofolate","EP_Vehicle_folate_1":"161171.709","EP_Vehicle_folate_2":"137400.074","EP_Vehicle_folate_3":"140644.706","EP_SHIN1_folate_1":"103179.794","EP_SHIN1_folate_2":"81368.2934","EP_SHIN1_folate_3":"91220.7011","EP_Inosine_folate_1":"151373.917","EP_Inosine_folate_2":"121432.944","EP_Inosine_folate_3":"134154.499","EP_SHIN1_Inosine_folate_1":"93862.3027","EP_SHIN1_Inosine_folate_2":"76055.8534","EP_SHIN1_Inosine_folate_3":"93658.032"},{"Metabolite":"5,10-methylenetetrahydrofolate","EP_Vehicle_folate_1":"360115.416","EP_Vehicle_folate_2":"325878.597","EP_Vehicle_folate_3":"350988.259","EP_SHIN1_folate_1":"153690.327","EP_SHIN1_folate_2":"140368.139","EP_SHIN1_folate_3":"135719.96","EP_Inosine_folate_1":"403155.701","EP_Inosine_folate_2":"308782.449","EP_Inosine_folate_3":"242878.861","EP_SHIN1_Inosine_folate_1":"232081.344","EP_SHIN1_Inosine_folate_2":"254667.65","EP_SHIN1_Inosine_folate_3":"268475.869"}],

"Metabolites":[{"Metabolite":"5-methyltetrahydrofolate","Standardized name":"5-Methyltetrahydrofolic acid","Formula":"C20H25N7O6","Exact mass":"459.1866","Sub class":"Pterins"},{"Metabolite":"5,10-methylenetetrahydrofolate","Standardized name":"-","Formula":"-","Exact mass":"-","Sub class":"-"}]
}

}