#METABOLOMICS WORKBENCH amaynard_20231002_164705 DATATRACK_ID:4365 STUDY_ID:ST002992 ANALYSIS_ID:AN004912 PROJECT_ID:PR001773 VERSION 1 CREATED_ON October 2, 2023, 5:07 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 Polar metabolite levels in K562 cells following C13-Serine and C13-Glycine ST:STUDY_TITLE tracing ST:STUDY_SUMMARY Culture of K562 cells for 7 days in RPMI media containing 2000 nM folic acid or ST:STUDY_SUMMARY 100 nM folic acid. At day 8, media was changed to 2000 nM or 100 nM folic acid ST:STUDY_SUMMARY with unlabeled serine and glycine, or 2-C13-Serine and 2-C13-Glycine at RPMI ST:STUDY_SUMMARY levels. Amino acid tracing was performed for 24 hours. These samples were ST:STUDY_SUMMARY followed up by metabolomics targeting polar 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 6 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS K-562 #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 K562 K562_2000nM_FA_Unlabeled_HILIC_1 Treatment:2000nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM447.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_Unlabeled_HILIC_2 Treatment:2000nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM448.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_Unlabeled_HILIC_3 Treatment:2000nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM449.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_Unlabeled_HILIC_4 Treatment:2000nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM450.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_Unlabeled_HILIC_1 Treatment:100nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM451.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_Unlabeled_HILIC_2 Treatment:100nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM452.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_Unlabeled_HILIC_3 Treatment:100nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM453.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_Unlabeled_HILIC_4 Treatment:100nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM454.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Glycine_HILIC_1 Treatment:2000nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM455.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Glycine_HILIC_2 Treatment:2000nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM456.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Glycine_HILIC_3 Treatment:2000nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM457.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Glycine_HILIC_4 Treatment:2000nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM458.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Serine_HILIC_1 Treatment:2000nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM459.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Serine_HILIC_2 Treatment:2000nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM460.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Serine_HILIC_3 Treatment:2000nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM461.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Serine_HILIC_4 Treatment:2000nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM462.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Glycine_HILIC_1 Treatment:100nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM467.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Glycine_HILIC_2 Treatment:100nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM468.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Glycine_HILIC_3 Treatment:100nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM469.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Glycine_HILIC_4 Treatment:100nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM470.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Serine_HILIC_1 Treatment:100nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM471.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Serine_HILIC_2 Treatment:100nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM472.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Serine_HILIC_3 Treatment:100nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM473.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Serine_HILIC_4 Treatment:100nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF1_HILIC_SGtracing_AM474.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 Culture of K562 cells for 7 days in RPMI media containing 2000 nM folic acid or TR:TREATMENT_SUMMARY 100 nM folic acid. At day 8, media was changed to 2000 nM or 100 nM folic acid TR:TREATMENT_SUMMARY with unlabeled serine and glycine, or 2-C13-Serine and 2-C13-Glycine at RPMI TR:TREATMENT_SUMMARY levels. Amino acid tracing was performed for 24 hours. The level of serine and TR:TREATMENT_SUMMARY glycine in all conditions was 30 and 10 mg/L, respectively. #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 polar metabolite detection. Dried samples were resuspended SP:SAMPLEPREP_SUMMARY in 25 μL water #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Sample was injected into a ZIC-pHILIC 150 x 2.1 mm (5 μm particle size) column CH:CHROMATOGRAPHY_SUMMARY (EMD Millipore). operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher CH:CHROMATOGRAPHY_SUMMARY Scientific, San Jose, CA, USA). Chromatographic separation was achieved using CH:CHROMATOGRAPHY_SUMMARY the following conditions: buffer A was acetonitrile; buffer B was 20 mM ammonium CH:CHROMATOGRAPHY_SUMMARY carbonate, 0.1% ammonium hydroxide in water; resulting pH is around 9 without pH CH:CHROMATOGRAPHY_SUMMARY adjustment. Gradient conditions we used were: linear gradient from 20% to 80% B; CH:CHROMATOGRAPHY_SUMMARY 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min CH:CHROMATOGRAPHY_SUMMARY flow rate. The column oven and autosampler tray were held at 25 °C and 4 °C, CH:CHROMATOGRAPHY_SUMMARY respectively. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME EMD Millipore ZIC-HILIC (100 x 2.1 mm,3.5 um) CH:SOLVENT_A 100% acetonitrile CH:SOLVENT_B 100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide CH:FLOW_GRADIENT linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 CH:FLOW_GRADIENT min: hold at 20% B at 150 mL/min flow rate CH:FLOW_RATE 150 mL/min CH:COLUMN_TEMPERATURE 25 #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 Polar metabolites were relatively quantified while referencing an in-house MS:MS_COMMENTS library of chemical standards and using TraceFinder 4.1 (Thermo Fisher MS:MS_COMMENTS Scientific, Waltham, MA, USA), with a 5 ppm mass tolerance. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples K562_2000nM_FA_Unlabeled_HILIC_1 K562_2000nM_FA_Unlabeled_HILIC_2 K562_2000nM_FA_Unlabeled_HILIC_3 K562_2000nM_FA_Unlabeled_HILIC_4 K562_100nM_FA_Unlabeled_HILIC_1 K562_100nM_FA_Unlabeled_HILIC_2 K562_100nM_FA_Unlabeled_HILIC_3 K562_100nM_FA_Unlabeled_HILIC_4 K562_2000nM_FA_C13_Glycine_HILIC_1 K562_2000nM_FA_C13_Glycine_HILIC_2 K562_2000nM_FA_C13_Glycine_HILIC_3 K562_2000nM_FA_C13_Glycine_HILIC_4 K562_2000nM_FA_C13_Serine_HILIC_1 K562_2000nM_FA_C13_Serine_HILIC_2 K562_2000nM_FA_C13_Serine_HILIC_3 K562_2000nM_FA_C13_Serine_HILIC_4 K562_100nM_FA_C13_Glycine_HILIC_1 K562_100nM_FA_C13_Glycine_HILIC_2 K562_100nM_FA_C13_Glycine_HILIC_3 K562_100nM_FA_C13_Glycine_HILIC_4 K562_100nM_FA_C13_Serine_HILIC_1 K562_100nM_FA_C13_Serine_HILIC_2 K562_100nM_FA_C13_Serine_HILIC_3 K562_100nM_FA_C13_Serine_HILIC_4 Factors Treatment:2000nM_FA_Unlabeled Treatment:2000nM_FA_Unlabeled Treatment:2000nM_FA_Unlabeled Treatment:2000nM_FA_Unlabeled Treatment:100nM_FA_Unlabeled Treatment:100nM_FA_Unlabeled Treatment:100nM_FA_Unlabeled Treatment:100nM_FA_Unlabeled Treatment:2000nM_FA_C13_Glycine Treatment:2000nM_FA_C13_Glycine Treatment:2000nM_FA_C13_Glycine Treatment:2000nM_FA_C13_Glycine Treatment:2000nM_FA_C13_Serine Treatment:2000nM_FA_C13_Serine Treatment:2000nM_FA_C13_Serine Treatment:2000nM_FA_C13_Serine Treatment:100nM_FA_C13_Glycine Treatment:100nM_FA_C13_Glycine Treatment:100nM_FA_C13_Glycine Treatment:100nM_FA_C13_Glycine Treatment:100nM_FA_C13_Serine Treatment:100nM_FA_C13_Serine Treatment:100nM_FA_C13_Serine Treatment:100nM_FA_C13_Serine glycine_C0 28910945.26 28323568.93 27571245.91 27065018.43 25190836 27014723.83 23604673.07 22953418.25 21816207.58 19008551.77 22771368.28 20427261.9 15088200.61 17822057.66 15469104.78 16345976.16 16786035.4 18731987.73 20484264.62 15772562.88 15880532.59 14576885.27 15152088.72 14812292.91 glycine_C1 195658.7077 138845.2247 115086.6486 680209.7138 304030.6308 602041.4227 449411.3482 380517.2598 7218146.3 6766689.128 6784921.782 6403520.731 15791003.97 17063561.49 16985904.84 17620964.99 8020342.687 8625825.808 9616263.459 4511892.886 10667473.56 9611142.869 10040128.8 9376719.967 serine_C0 23728906.68 20714183.64 18761904.75 20210119.21 43001237.82 44647401.71 41002145.67 42874227.12 19436809.2 17517434.16 20157826.69 17996333.57 12531166.75 12725181.23 13228048.54 16925803.27 38952767.76 40460591.25 45012678.05 75282338.96 23240876.77 21887183.99 22570229.68 22988202.26 serine_C1 426134.5593 320826.1607 292344.8723 288268.4807 1085644.903 1322206.413 845890.738 1290215.339 1969774.641 2002676.178 1505230.784 1722314.939 7035208.931 7593615.542 6980693.515 7449906.862 3668391.027 3991050.416 4268807.998 3727105.622 21331787.85 19522694.32 19712834.15 19260639.09 IMP_C0 1624064.619 1540143.995 1527395.903 1639995.792 1600550.404 1571738.114 1575379.802 1597515.972 1462067.032 1459186.867 1610870.85 1437302.782 943621.6398 1039919.984 927295.1245 879549.1899 1222315.025 1189315.799 1408546.96 646655.6449 1470822.4 1393099.764 1202996.795 1258255.801 IMP_C1 36307.21821 59088.1903 63001.89252 63071.82729 36780.08874 33387.62271 65428.25114 58856.6787 409321.575 497981.9764 553299.4601 448969.0259 847433.7114 1032527.176 955941.0272 947564.3104 455806.6961 513209.5491 519310.8276 209084.0391 668725.2998 432927.9241 431625.6494 474426.4423 GSSG_C0 7371040.954 5669224.773 7207107.816 7032687.222 5466351.815 5818343.124 4791332.872 3820397.512 6079203.414 5171375.516 15090343.39 4336154.45 452764.9423 1086200.663 2307241.096 1302915.992 2435470.304 2467036.005 1746999.666 954744.1982 5113615.197 8178344.528 3333337.624 3448148.125 GSSG_C1 1779464.637 1408653.972 1867594.154 1744258.164 1428120.289 1494242.724 1208976.804 949593.3902 5073699.884 4348276.129 12519887.24 3403501.827 1602381.443 3391443.953 7174069.466 4144105.421 3135042.59 3282297.625 2251122.759 1262052.279 8488933.945 13220469.14 5566163.853 5783740.823 GSSG_C2 0 0 26390.6293 38957.95654 84357.55641 0 38438.79807 27838.5867 1480463.14 1363174.869 3605314.084 911080.7767 1290932.577 2931998.77 6223691.227 3875879.28 1303257.406 1413169.584 809417.3979 457628.8694 4163477.4 6531076.784 2694361.083 2755713.562 GSH_Ellmans_C0 317706249.4 325683954.7 321884459.2 334877619.3 270263986.8 266916595.5 275272019 287989333.3 239637362.2 265878578.6 217113669.2 278010717.1 127224459.2 145534639.9 133860383.3 131691510.6 189875562.8 176385295.4 179366255 86349949.87 163156999.3 151526385.6 141285561.4 184643693.7 GSH_Ellmans_C1 66293958.68 68370774.08 66386541.43 71065984.99 55145778.34 54666512.04 56848786.64 58412014.39 122065100.2 136482712.8 110212105.3 141384298.8 214527375.1 246012587.6 224296690.1 221366258.1 145555671 136948009.7 138942489.2 66251025.2 150603585.6 141177644.3 129020252.2 165007048 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name glycine_C0 glycine_C1 serine_C0 serine_C1 IMP_C0 IMP_C1 GSSG_C0 GSSG_C1 GSSG_C2 GSH_Ellmans_C0 GSH_Ellmans_C1 METABOLITES_END #END