#METABOLOMICS WORKBENCH amaynard_20231219_200831 DATATRACK_ID:4543 STUDY_ID:ST003023 ANALYSIS_ID:AN004957 PROJECT_ID:PR001773 VERSION 1 CREATED_ON December 19, 2023, 8:12 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 metabolite levels in K562 cells following 6 day culture in 2,000 nM or ST:STUDY_TITLE 100 nM folic acid ST:STUDY_SUMMARY Culture of K562 cells in RPMI media containing 2000 nM or 100 nM folic acid for ST:STUDY_SUMMARY 6 days. These samples were followed up by metabolomics targeting porphyrin ST:STUDY_SUMMARY 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_Porphyrin_1 Folic_Acid_Concentration:2000_nM RAW_FILE_NAME=Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM278.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_Porphyrin_2 Folic_Acid_Concentration:2000_nM RAW_FILE_NAME=Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM279.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_Porphyrin_3 Folic_Acid_Concentration:2000_nM RAW_FILE_NAME=Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM280.raw SUBJECT_SAMPLE_FACTORS K562 K562_2000nM_Porphyrin_4 Folic_Acid_Concentration:2000_nM RAW_FILE_NAME=Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM281.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Porpyrin_1 Folic_Acid_Concentration:100_nM RAW_FILE_NAME=Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM282.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Porpyrin_2 Folic_Acid_Concentration:100_nM RAW_FILE_NAME=Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM283.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Porpyrin_3 Folic_Acid_Concentration:100_nM RAW_FILE_NAME=Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM284.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Porpyrin_4 Folic_Acid_Concentration:100_nM RAW_FILE_NAME=Copy of 20210201_QEF2_C18_HemeAdamC6KOs_AM285.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 6 days in RPMI media containing 2000 nM or 100 nM TR:TREATMENT_SUMMARY folic acid for 6 days. These samples were followed up by metabolomics targeting TR:TREATMENT_SUMMARY porphyrin metabolites. #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 Arbitrary Units MS_METABOLITE_DATA_START Samples K562_2000nM_Porphyrin_1 K562_2000nM_Porphyrin_2 K562_2000nM_Porphyrin_3 K562_2000nM_Porphyrin_4 K562_100nM_Porpyrin_1 K562_100nM_Porpyrin_2 K562_100nM_Porpyrin_3 K562_100nM_Porpyrin_4 Factors Folic_Acid_Concentration:2000_nM Folic_Acid_Concentration:2000_nM Folic_Acid_Concentration:2000_nM Folic_Acid_Concentration:2000_nM Folic_Acid_Concentration:100_nM Folic_Acid_Concentration:100_nM Folic_Acid_Concentration:100_nM Folic_Acid_Concentration:100_nM Biliverdin 190999.5034 277219.3206 213790.6119 228208.118 256222.7501 280173.5827 227497.6816 254174.1875 Heme 15060580.3 12080916.52 15723822.73 13846413.2 69215619.06 76706810.94 67696074.88 74083933.98 Protoporphyrin IX 24415.38995 32860.48443 40204.07607 23893.14971 116093.4279 129099.3213 98061.14129 115579.2699 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Formula Exact mass Sub class Biliverdin Biliverdin C33H34N4O6 582.2478 Bilirubins Heme Heme C34H32FeN4O4 616.1773 Metalloporphyrins Protoporphyrin IX Protoporphyrin C34H34N4O4 562.2580 Porphyrins METABOLITES_END #END