#METABOLOMICS WORKBENCH amaynard_20230928_194129 DATATRACK_ID:4341 STUDY_ID:ST002893 ANALYSIS_ID:AN004752 PROJECT_ID:PR001773 VERSION 1 CREATED_ON October 2, 2023, 8:47 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 Folate depletion time-course in MEL cells with analysis for porphyrin ST:STUDY_TITLE metabolites ST:STUDY_SUMMARY Culture of MEL cells in RPMI media containing 100 nM folic acid for 0, 1, 2, 4, ST:STUDY_SUMMARY 6, or 8 days followed my LC-MS targeting porphyrin metabolites. This is a ST:STUDY_SUMMARY reverse timecourse where all samples are harvested on the same day. Day 0 in 100 ST:STUDY_SUMMARY nM folic acid indicates 8 days culture in 2,000 nM folic acid. 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 Mus musculus SU:TAXONOMY_ID 10090 SU:CELL_STRAIN_DETAILS MEL #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 MEL MEL_100nM_Timecourse_Day0_Porphyrin_1 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM691.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day0_Porphyrin_2 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM692.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day0_Porphyrin_3 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM693.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day1_Porphyrin_1 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM694.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day1_Porphyrin_2 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM695.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day1_Porphyrin_3 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM696.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day1_Porphyrin_4 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM697.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day2_Porphyrin_1 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM698.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day2_Porphyrin_2 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM699.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day2_Porphyrin_3 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM700.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day2_Porphyrin_4 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM701.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day4_Porphyrin_1 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM702.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day4_Porphyrin_2 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM703.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day4_Porphyrin_3 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM704.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day4_Porphyrin_4 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM705.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day6_Porphyrin_1 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM706.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day6_Porphyrin_2 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM707.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day6_Porphyrin_3 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM708.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day8_Porphyrin_1 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM709.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day8_Porphyrin_2 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM710.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day8_Porphyrin_3 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20220405_AM_HEME_MEL_Timecourse_AM711.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 MEL cells were cultured for 0, 1, 2, 4, or 6 days in 100 nM folic acid TR:TREATMENT_SUMMARY containing RPMI media. #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% 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 peak area MS_METABOLITE_DATA_START Samples MEL_100nM_Timecourse_Day0_Porphyrin_1 MEL_100nM_Timecourse_Day0_Porphyrin_2 MEL_100nM_Timecourse_Day0_Porphyrin_3 MEL_100nM_Timecourse_Day1_Porphyrin_1 MEL_100nM_Timecourse_Day1_Porphyrin_2 MEL_100nM_Timecourse_Day1_Porphyrin_3 MEL_100nM_Timecourse_Day1_Porphyrin_4 MEL_100nM_Timecourse_Day2_Porphyrin_1 MEL_100nM_Timecourse_Day2_Porphyrin_2 MEL_100nM_Timecourse_Day2_Porphyrin_3 MEL_100nM_Timecourse_Day2_Porphyrin_4 MEL_100nM_Timecourse_Day4_Porphyrin_1 MEL_100nM_Timecourse_Day4_Porphyrin_2 MEL_100nM_Timecourse_Day4_Porphyrin_3 MEL_100nM_Timecourse_Day4_Porphyrin_4 MEL_100nM_Timecourse_Day6_Porphyrin_1 MEL_100nM_Timecourse_Day6_Porphyrin_2 MEL_100nM_Timecourse_Day6_Porphyrin_3 MEL_100nM_Timecourse_Day8_Porphyrin_1 MEL_100nM_Timecourse_Day8_Porphyrin_2 MEL_100nM_Timecourse_Day8_Porphyrin_3 Factors 100nM_Folic_Acid_Growth:Day0 100nM_Folic_Acid_Growth:Day0 100nM_Folic_Acid_Growth:Day0 100nM_Folic_Acid_Growth:Day1 100nM_Folic_Acid_Growth:Day1 100nM_Folic_Acid_Growth:Day1 100nM_Folic_Acid_Growth:Day1 100nM_Folic_Acid_Growth:Day2 100nM_Folic_Acid_Growth:Day2 100nM_Folic_Acid_Growth:Day2 100nM_Folic_Acid_Growth:Day2 100nM_Folic_Acid_Growth:Day4 100nM_Folic_Acid_Growth:Day4 100nM_Folic_Acid_Growth:Day4 100nM_Folic_Acid_Growth:Day4 100nM_Folic_Acid_Growth:Day6 100nM_Folic_Acid_Growth:Day6 100nM_Folic_Acid_Growth:Day6 100nM_Folic_Acid_Growth:Day8 100nM_Folic_Acid_Growth:Day8 100nM_Folic_Acid_Growth:Day8 Heme 863520.8 1418628 995986.7 4725143 7562818 3088104 5433601 8433923 6467621 4377637 5593699 4025685 629455.1 334381.4 6474829 13646083 16219467 5645590 19729657 22808196 11212765 Protoporphyrin IX 1139539 1177029 470946.4 1151470 819424.4 751937.3 559192.4 481386.1 438233.3 795875.1 217574.5 795183 425237 433941 291813.5 1492499 713975.1 417354.4 752363.4 832947.1 867931.1 Biliverdin 3434.348 4384.505 3077.536 1271882 3830990 25438.96 1817412 2413254 1831494 1468973 1937738 27966.87 0 599.9179 246389 1658960 1577056 0 1213552 1583033 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Formula Exact mass Sub class Heme - C34H32FeN4O4 616.1773 Porphyrins Protoporphyrin IX Protoporphyrin C34H34N4O4 562.258 Porphyrins Biliverdin Biliverdin C33H34N4O6 582.2478 Bilirubins METABOLITES_END #END