#METABOLOMICS WORKBENCH amaynard_20231219_193057 DATATRACK_ID:4540 STUDY_ID:ST003021 ANALYSIS_ID:AN004955 PROJECT_ID:PR001773 VERSION 1 CREATED_ON December 21, 2023, 10:02 am #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 PKC inhibition in 2,000 nM ST:STUDY_TITLE or 100 nM FA conditions ST:STUDY_SUMMARY Culture of K562 cells in RPMI media containing 2000 nM or 100 nM folic acid for ST:STUDY_SUMMARY 6 days in the presence or absence of the PKC inhibitor, GF109203X. These samples ST:STUDY_SUMMARY were 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_longterm_PKCi_Porphyrin_2000nM_FA_1 Treatment:2000nM_FA_Veh RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1105.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_2000nM_FA_2 Treatment:2000nM_FA_Veh RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1106.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_2000nM_FA_3 Treatment:2000nM_FA_Veh RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1107.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_2000nM_FA_4 Treatment:2000nM_FA_Veh RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1108.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_1 Treatment:2000nM_FA_GFX RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1113.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_2 Treatment:2000nM_FA_GFX RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1114.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_3 Treatment:2000nM_FA_GFX RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1115.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_4 Treatment:2000nM_FA_GFX RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1116.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_100nM_FA_1 Treatment:100nM_FA_Veh RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1117.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_100nM_FA_2 Treatment:100nM_FA_Veh RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1118.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_100nM_FA_3 Treatment:100nM_FA_Veh RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1119.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_100nM_FA_4 Treatment:100nM_FA_Veh RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1120.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_1 Treatment:100nM_FA_GFX RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1125.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_2 Treatment:100nM_FA_GFX RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1126.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_3 Treatment:100nM_FA_GFX RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1127.zip SUBJECT_SAMPLE_FACTORS K562 K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_4 Treatment:100nM_FA_GFX RAW_FILE_NAME=Copy of 20231006_QEF2_Phenomenex_K562Heme_AdamRevision_AM1128.zip #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 in the presence or absence of the PKC inhibitor, TR:TREATMENT_SUMMARY GF109203X. These samples were followed up by metabolomics targeting polar TR:TREATMENT_SUMMARY 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 0–2 min: 5% B; 2-19min: linear gradient from 5% to 95% B; 19-21min: 95% B; CH:FLOW_GRADIENT 21.1-23min: return to 5% B. 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_longterm_PKCi_Porphyrin_2000nM_FA_1 K562_longterm_PKCi_Porphyrin_2000nM_FA_2 K562_longterm_PKCi_Porphyrin_2000nM_FA_3 K562_longterm_PKCi_Porphyrin_2000nM_FA_4 K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_1 K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_2 K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_3 K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_4 K562_longterm_PKCi_Porphyrin_100nM_FA_1 K562_longterm_PKCi_Porphyrin_100nM_FA_2 K562_longterm_PKCi_Porphyrin_100nM_FA_3 K562_longterm_PKCi_Porphyrin_100nM_FA_4 K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_1 K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_2 K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_3 K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_4 Factors Treatment:2000nM_FA_Veh Treatment:2000nM_FA_Veh Treatment:2000nM_FA_Veh Treatment:2000nM_FA_Veh Treatment:2000nM_FA_GFX Treatment:2000nM_FA_GFX Treatment:2000nM_FA_GFX Treatment:2000nM_FA_GFX Treatment:100nM_FA_Veh Treatment:100nM_FA_Veh Treatment:100nM_FA_Veh Treatment:100nM_FA_Veh Treatment:100nM_FA_GFX Treatment:100nM_FA_GFX Treatment:100nM_FA_GFX Treatment:100nM_FA_GFX Protoporphyrin IX 828411.5585 1172081.197 1105617.563 986724.4185 1655479.154 1396615.852 980616.1505 1994110.825 666588.1733 784178.1367 597023.4338 697310.0035 1413512.347 1558641.737 1349411.788 2034189.117 Heme 10788228.38 19157735.88 18546287.64 19452133.69 30455088.2 32367013.5 23848805.37 33180707.44 54476342.49 53996897.75 42991445.71 58563550.92 38749333.32 42515189.81 34831040.03 61916755.63 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Formula Exact mass Sub class Protoporphyrin IX Protoporphyrin C34H34N4O4 562.2580 Porphyrins Heme Heme C34H32FeN4O4 616.1773 Metalloporphyrins METABOLITES_END #END