#METABOLOMICS WORKBENCH amaynard_20231001_210302 DATATRACK_ID:4360 STUDY_ID:ST002900 ANALYSIS_ID:AN004759 PROJECT_ID:PR001773 VERSION 1 CREATED_ON October 1, 2023, 9:09 pm #PROJECT PR:PROJECT_TITLE Folate depletion induces erythroid differentiation through perturbation of de PR:PROJECT_TITLE novo purine synthesis PR:PROJECT_SUMMARY Folate, an essential vitamin, is a one-carbon acceptor and donor in key PR:PROJECT_SUMMARY metabolic reactions. Erythroid cells harbor a unique sensitivity to folate PR:PROJECT_SUMMARY deprivation, as revealed by the primary pathological manifestation of PR:PROJECT_SUMMARY nutritional folate deprivation: megaloblastic anemia. To study this metabolic PR:PROJECT_SUMMARY sensitivity, we applied mild folate depletion to human and mouse erythroid cell PR:PROJECT_SUMMARY lines, and primary murine erythroid progenitors. We show that folate depletion PR:PROJECT_SUMMARY induces early blockade of purine synthesis and accumulation of the purine PR:PROJECT_SUMMARY synthesis intermediate and signaling molecule, AICAR, followed by enhanced heme PR:PROJECT_SUMMARY metabolism, hemoglobin synthesis, and erythroid differentiation. This is PR:PROJECT_SUMMARY phenocopied by inhibition of folate metabolism using the SHMT1/2 inhibitor - PR:PROJECT_SUMMARY SHIN1, and by AICAR supplementation. Mechanistically, the metabolically-driven PR:PROJECT_SUMMARY differentiation is independent of nucleotide sensing through mTORC1 and AMPK, PR:PROJECT_SUMMARY and is instead mediated by protein kinase C (PKC). Our findings suggest that PR:PROJECT_SUMMARY folate deprivation-induced premature differentiation of erythroid progenitor PR:PROJECT_SUMMARY cells is a molecular etiology to folate-deficiency induced anemia. PR:INSTITUTE Boston Children's Hospital, Harvard Medical School PR:DEPARTMENT pathology PR:LABORATORY Kanarek Lab PR:LAST_NAME Kanarek PR:FIRST_NAME Naama PR:ADDRESS Enders 1116.2, 300 Longwood Ave, Boston, MA 02115 PR:EMAIL naama.kanarek@childrens.harvard.edu PR:PHONE (617) 355-7433 #STUDY ST:STUDY_TITLE Folate metabolite levels in erythroid progenitor cells following SHIN1 and ST:STUDY_TITLE inosine treatment ST:STUDY_SUMMARY Culture of erythroid progenitor cells cultured in SFEM II media supplemented ST:STUDY_SUMMARY with SHIN1 and/or inosine for 48 hours followed up by metabolomics targeting ST:STUDY_SUMMARY folate metabolites. ST:INSTITUTE Boston Children's Hospital, Harvard Medical School ST:DEPARTMENT pathology ST:LABORATORY Kanarek Lab ST:LAST_NAME Kanarek ST:FIRST_NAME Naama ST:ADDRESS Enders 1116.2, 300 Longwood Ave, Boston, MA 02115 ST:EMAIL naama.kanarek@childrens.harvard.edu ST:PHONE 6173557433 ST:NUM_GROUPS 4 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:CELL_BIOSOURCE_OR_SUPPLIER Lineage depleted erythroid progenitor cells isolated from E14.5 mice. #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_Vehicle_folate_1 Treatment:Vehicle RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM916.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_Vehicle_folate_2 Treatment:Vehicle RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM917.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_Vehicle_folate_3 Treatment:Vehicle RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM918.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_SHIN1_folate_1 Treatment:SHIN1 RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM919.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_SHIN1_folate_2 Treatment:SHIN1 RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM920.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_SHIN1_folate_3 Treatment:SHIN1 RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM921.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_Inosine_folate_1 Treatment:Inosine RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM922.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_Inosine_folate_2 Treatment:Inosine RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM923.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_Inosine_folate_3 Treatment:Inosine RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM924.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_SHIN1_Inosine_folate_1 Treatment:SHIN1_Inosine RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM925.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_SHIN1_Inosine_folate_2 Treatment:SHIN1_Inosine RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM926.raw SUBJECT_SAMPLE_FACTORS Erythroid_Progenitor EP_SHIN1_Inosine_folate_3 Treatment:SHIN1_Inosine RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM927.raw #COLLECTION CO:COLLECTION_SUMMARY One million cells from culture were collected via centrifugation for 20 seconds CO:COLLECTION_SUMMARY at 18,000xG, washed with 0.9% NaCl, and collected via centrifugation for 20 CO:COLLECTION_SUMMARY seconds at 18,000xG CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Erythroid progenitor cells were cultured for 48hr in SFEM II media containing TR:TREATMENT_SUMMARY vehicle, SHIN1 (1.25 uM), inosine (100 uM), or SHIN1+inosine. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Pellet was resuspended in extraction buffer (80% Methanol, 25 mM Ammonium SP:SAMPLEPREP_SUMMARY Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with SP:SAMPLEPREP_SUMMARY isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, SP:SAMPLEPREP_SUMMARY MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope SP:SAMPLEPREP_SUMMARY Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged SP:SAMPLEPREP_SUMMARY for 10 minutes at 18,000 g to pellet cell debris. The supernatant was divided SP:SAMPLEPREP_SUMMARY into two tubes and dried on ice using a liquid nitrogen dryer. One tube of dried SP:SAMPLEPREP_SUMMARY sample was used for folate metabolite detection. Folate samples were resuspended SP:SAMPLEPREP_SUMMARY in reconstitution buffer (0.5% ascorbic acid, 1% K2HPO4 and 0.5% SP:SAMPLEPREP_SUMMARY 2-mercaptoethanol) containing rat serum (Sigma, R9759). The rat serum contains SP:SAMPLEPREP_SUMMARY the enzymes required to strip the polyglutamate tail from the measured folate. SP:SAMPLEPREP_SUMMARY Rat endogenous folate was removed from the rat serum by activated carbon SP:SAMPLEPREP_SUMMARY treatment (Sigma, C9157). Polyglutamate tail removal was carried out for 2 h SP:SAMPLEPREP_SUMMARY at 37 °C. After the polyglutamate tail removal, the pH was adjusted to 4 SP:SAMPLEPREP_SUMMARY using formic acid and samples were loaded on Bond Elute-pH columns (Agilent, SP:SAMPLEPREP_SUMMARY 14102062) for a pH-based clean-up. Columns were eluted with 300 μl elution SP:SAMPLEPREP_SUMMARY buffer (50% methanol, 0.25% NH4OAc, 0.5% 2-mercaptoethanol), followed by drying SP:SAMPLEPREP_SUMMARY the samples using a liquid nitrogen dryer manifold and resuspension in 25 μl SP:SAMPLEPREP_SUMMARY water. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 5 μl was injected onto a 2.6 μm, 150 x 3 mm C18 column (Phenomenex, CH:CHROMATOGRAPHY_SUMMARY 00F-4462-Y0) equipped with a 3.0 mm safe-guard column (Phenomenex, AJ0-8775). CH:CHROMATOGRAPHY_SUMMARY The column oven and autosampler tray were held at 30 °C and 4 °C, CH:CHROMATOGRAPHY_SUMMARY respectively. The following conditions were used to achieve chromatographic CH:CHROMATOGRAPHY_SUMMARY separation: buffer A was 0.1% formic acid; buffer B was acetonitrile with 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid. The chromatographic gradient was run at a flow rate of 0.250 ml CH:CHROMATOGRAPHY_SUMMARY min−1 as follows: 0–5 min: gradient was held at 5% B; 5–10 min: linear CH:CHROMATOGRAPHY_SUMMARY gradient of 5% to 36% B; 10.1–14.0 min: linear gradient from 36–95% B; CH:CHROMATOGRAPHY_SUMMARY 14.1–18.0 min: gradient was returned to 5% B. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) CH:SOLVENT_A 0.1% formic acid CH:SOLVENT_B acetonitrile with 0.1% formic acid CH:FLOW_GRADIENT 0.250 ml min−1 as follows: 0–5 min: gradient was held at 5% B; CH:FLOW_GRADIENT 5–10 min: linear gradient of 5% to 36% B; 10.1–14.0 min: linear gradient CH:FLOW_GRADIENT from 36–95% B; 14.1–18.0 min: gradient was returned to 5% B. CH:FLOW_RATE 250 uL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS The mass spectrometer was operated in full-scan, positive ionization mode using MS:MS_COMMENTS three narrow-range scans: 438–450 m/z; 452–462 m/z; and 470–478 m/z, with MS:MS_COMMENTS the resolution set at 70,000, the AGC target at 10e6, and the maximum injection MS:MS_COMMENTS time of 150 ms. HESI settings were: sheath gas flow rate: 40; Aux gas flow MS:MS_COMMENTS rate: 10; Sweep gas: 0; Spray voltage: 2.8 (neg) 3.5 (pos); Capillary MS:MS_COMMENTS temperature 300; S-lens RF level 50; Aux gas heater temp: 350. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples EP_Vehicle_folate_1 EP_Vehicle_folate_2 EP_Vehicle_folate_3 EP_SHIN1_folate_1 EP_SHIN1_folate_2 EP_SHIN1_folate_3 EP_Inosine_folate_1 EP_Inosine_folate_2 EP_Inosine_folate_3 EP_SHIN1_Inosine_folate_1 EP_SHIN1_Inosine_folate_2 EP_SHIN1_Inosine_folate_3 Factors Treatment:Vehicle Treatment:Vehicle Treatment:Vehicle Treatment:SHIN1 Treatment:SHIN1 Treatment:SHIN1 Treatment:Inosine Treatment:Inosine Treatment:Inosine Treatment:SHIN1_Inosine Treatment:SHIN1_Inosine Treatment:SHIN1_Inosine 5-methyltetrahydrofolate 161171.709 137400.074 140644.706 103179.794 81368.2934 91220.7011 151373.917 121432.944 134154.499 93862.3027 76055.8534 93658.032 5,10-methylenetetrahydrofolate 360115.416 325878.597 350988.259 153690.327 140368.139 135719.96 403155.701 308782.449 242878.861 232081.344 254667.65 268475.869 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Formula Exact mass Sub class 5-methyltetrahydrofolate 5-Methyltetrahydrofolic acid C20H25N7O6 459.1866 Pterins 5,10-methylenetetrahydrofolate - - - - METABOLITES_END #END