#METABOLOMICS WORKBENCH amaynard_20231002_173316 DATATRACK_ID:4366 STUDY_ID:ST002905 ANALYSIS_ID:AN004768 PROJECT_ID:PR001773 VERSION 1 CREATED_ON October 2, 2023, 5:45 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 Porphyrin analysis of K562 cells following C13-Serine and C13-Glycine tracing ST:STUDY_TITLE during folate depletion. 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 porphyrin 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 5 #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_porphyrin_1 Treatment:2000nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM506.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_Unlabeled_porphyrin_2 Treatment:2000nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM507.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_Unlabeled_porphyrin_3 Treatment:2000nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM508.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_Unlabeled_porphyrin_4 Treatment:2000nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM509.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_Unlabeled_porphyrin_1 Treatment:100nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM510.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_Unlabeled_porphyrin_2 Treatment:100nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM511.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_Unlabeled_porphyrin_3 Treatment:100nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM512.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_Unlabeled_porphyrin_4 Treatment:100nM_FA_Unlabeled RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM513.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Glycine_porphyrin_1 Treatment:2000nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM514.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Glycine_porphyrin_2 Treatment:2000nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM515.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Glycine_porphyrin_3 Treatment:2000nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM516.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Glycine_porphyrin_4 Treatment:2000nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM517.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Serine_porphyrin_1 Treatment:2000nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM518.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Serine_porphyrin_2 Treatment:2000nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM519.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Serine_porphyrin_3 Treatment:2000nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM520.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_FA_C13_Serine_porphyrin_4 Treatment:2000nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM521.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Glycine_porphyrin_1 Treatment:100nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM526.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Glycine_porphyrin_2 Treatment:100nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM527.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Glycine_porphyrin_3 Treatment:100nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM528.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Glycine_porphyrin_4 Treatment:100nM_FA_C13_Glycine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM529.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Serine_porphyrin_1 Treatment:100nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM530.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Serine_porphyrin_2 Treatment:100nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM531.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Serine_porphyrin_3 Treatment:100nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM532.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_FA_C13_Serine_porphyrin_4 Treatment:100nM_FA_C13_Serine RAW_FILE_NAME=Copy of 20210903_QEF2_C18_SGTracing_AM533.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 One million cells from culture were collected via centrifugation, washed with SP:SAMPLEPREP_SUMMARY 0.9% NaCl, and resuspended in 150 µl of porphyrin extraction buffer (1:4 ratio SP:SAMPLEPREP_SUMMARY of 1.7 M HCl:ACN, 1µM deuteroporphyrin IX (Frontier Scientific, D510-9)) and SP:SAMPLEPREP_SUMMARY 0.5 µM isotopically labeled amino acids (Cambridge Isotopes, MSK-A2-1.2)). SP:SAMPLEPREP_SUMMARY Samples were vigorously shaken for 20 min at 16ºC in a thermomixer (Eppendorf), SP:SAMPLEPREP_SUMMARY sonicated for 10 cycles at 4ºC with 30 sec on and 30 sec off, then incubated at SP:SAMPLEPREP_SUMMARY 4ºC for 10 min. Following incubation on ice, samples were centrifuged for 10 SP:SAMPLEPREP_SUMMARY minutes at 18,000g to pellet cell debris. The supernatant was collected and 40.5 SP:SAMPLEPREP_SUMMARY µl super-saturated MgSO4 and 12 µl 5 M NaCl were added. Samples were vortexed SP:SAMPLEPREP_SUMMARY for 30 sec and further shaken for 10 min at 16 ºC in a thermomixer. Finally, a SP:SAMPLEPREP_SUMMARY 10 min 10,000 rpm centrifugation was used to separate the organic layer (upper) SP:SAMPLEPREP_SUMMARY from the aqueous layer (lower). The upper organic layer was collected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Sample (5 uL) 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 Column compartment was heated to 45 ºC. Porphyrins were separated with a CH:CHROMATOGRAPHY_SUMMARY chromatographic gradient at a flow rate of 0.800 ml min−1 as follows: CH:CHROMATOGRAPHY_SUMMARY 0–2 min: 5% B; 2-19min: linear gradient from 5% to 95% B; 19-21min: 95% B; CH:CHROMATOGRAPHY_SUMMARY 21.1-23min: return to 5% B. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 3mm,2.6um) CH:SOLVENT_A 95% water/5% acetonitrile; 0.1% formic acid CH:SOLVENT_B 5% water/95% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT linear gradient from 5% to 95% CH:FLOW_RATE 0.8 mL/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometer was operated in full-scan, positive ionization mode using MS:MS_COMMENTS a narrow-range scan: 450-700m/z, with an additional tSIM scan for hemin MS:MS_COMMENTS (616.1767 m/z), CoproP (655.2762 m/z), and PPIX (563.2653 m/z). Metabolites were MS:MS_COMMENTS relatively quantified while referencing an in-house library of chemical MS:MS_COMMENTS standards and using TraceFinder 4.1 (Thermo Fisher Scientific, Waltham, MA, MS:MS_COMMENTS 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_porphyrin_1 K562_2000nM_FA_Unlabeled_porphyrin_2 K562_2000nM_FA_Unlabeled_porphyrin_3 K562_2000nM_FA_Unlabeled_porphyrin_4 K562_100nM_FA_Unlabeled_porphyrin_1 K562_100nM_FA_Unlabeled_porphyrin_2 K562_100nM_FA_Unlabeled_porphyrin_3 K562_100nM_FA_Unlabeled_porphyrin_4 K562_2000nM_FA_C13_Glycine_porphyrin_1 K562_2000nM_FA_C13_Glycine_porphyrin_2 K562_2000nM_FA_C13_Glycine_porphyrin_3 K562_2000nM_FA_C13_Glycine_porphyrin_4 K562_2000nM_FA_C13_Serine_porphyrin_1 K562_2000nM_FA_C13_Serine_porphyrin_2 K562_2000nM_FA_C13_Serine_porphyrin_3 K562_2000nM_FA_C13_Serine_porphyrin_4 K562_100nM_FA_C13_Glycine_porphyrin_1 K562_100nM_FA_C13_Glycine_porphyrin_2 K562_100nM_FA_C13_Glycine_porphyrin_3 K562_100nM_FA_C13_Glycine_porphyrin_4 K562_100nM_FA_C13_Serine_porphyrin_1 K562_100nM_FA_C13_Serine_porphyrin_2 K562_100nM_FA_C13_Serine_porphyrin_3 K562_100nM_FA_C13_Serine_porphyrin_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 heme_C0 24860654.1 13561566.13 696461.1136 12865741.61 102937914.5 52690882.19 54016176.51 72572865963 11325636.74 15167597.43 13180984.41 8835307.597 3294057.848 9842062.88 3695771.746 2084378.507 172771229.7 201589194.5 196603133.8 102468577.6 41551626.42 124339594 167452623.6 92260049.28 heme_C1 10133354.54 5543460.095 284444.7111 5444066.789 42384056.92 21735058.64 22444411.42 30123310417 6292503.003 8383377.656 7172408.353 4793695.815 1279665.023 4202647.569 1469190.625 829274.0139 79538831.29 92685435.66 90276701.97 47494045.15 18643271.58 56205638.84 76137099.5 41828887.02 heme_C2 2144170.472 1149389.43 21019.82477 1077726.385 9181595.212 4653254.67 4850758.892 6565974293 3096717.401 4200171.93 3560250.086 2312787.157 324049.9596 1018150.503 369995.4403 195312.3775 32160549.42 35813604.96 35497920.15 18774722.51 6790555.333 21798901.85 27961299.1 15947566.97 heme_C3 308478.3031 171309.8735 0 159999.3265 1297344.7 657720.147 668095.9766 899724336.2 1641657.963 2422975.529 2005185.153 1199085.5 238368.3547 743103.2285 273935.2669 117938.0888 23462765.96 26796370.77 26094766.03 13606099.85 5116681.322 16141283.27 20879138.13 11767979.25 heme_C4 33597.27979 22624.21706 0 16520.60007 140071.9152 75380.42625 82695.45148 110426170.7 815585.8304 1108058.262 889383.6511 577280.9946 388373.1063 1299717.198 460822.8256 198500.1547 20254099.11 22976412.84 22046529.97 11762824.97 4699620.295 14802337.62 19776677.21 10795452.91 heme_C5 587.9445044 0 0 0 0 0 4340.621273 1924183.313 282556.6657 372238.4084 337736.6158 216691.2357 546877.2221 1762684.097 639632.9128 294752.1747 14159673.17 16142239.29 15740014.85 8237427.92 3447248.48 10726160.08 14016123.79 7764886.249 heme_C6 0 0 0 0 0 0 5292.986009 8795449.925 72436.78923 106665.3217 71754.7212 62298.2912 442386.0457 1629162.022 558842.3168 277604.882 7104653.865 8301934.461 8069737.048 4263417.725 1872588.054 5541774.594 7483109.244 4048611.319 heme_C7 0 0 0 0 0 0 0 46505098.29 4529.075634 24333.72929 2410.589564 28856.31356 261131.7319 934222.9941 291428.1838 174155.5909 2669341.287 3155726.425 3051201.355 1601306.269 672825.3469 2036968.081 2832773.864 1570996.774 heme_C8 0 0 0 0 0 0 0 32034279.4 0 1075.550957 0 10978.66354 89440.35619 312109.3406 106333.1926 130621.5268 675928.9161 795127.4188 748912.7185 410282.3822 157312.7443 518720.4931 696527.9615 437327.9599 heme_C9 0 0 0 0 0 0 0 0 0 0 0 0 0 68154.69223 0 0 414364.5084 463224.7831 574774.0746 264676.4044 27348.05832 177119.1826 436934.2228 239522.1358 heme_C10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 101995.1248 101809.4925 153987.8281 75352.96438 5775.132488 113764.8509 112154.2896 73244.44255 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name heme_C0 heme_C1 heme_C2 heme_C3 heme_C4 heme_C5 heme_C6 heme_C7 heme_C8 heme_C9 heme_C10 METABOLITES_END #END