#METABOLOMICS WORKBENCH amaynard_20231002_175921 DATATRACK_ID:4368 STUDY_ID:ST002907 ANALYSIS_ID:AN004770 PROJECT_ID:PR001773 VERSION 1 CREATED_ON October 2, 2023, 6:04 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 Folate levels in K562 cells following short-term folate depletion ST:STUDY_SUMMARY Culture of K562 cells for 48hr in RPMI media containing 2000 nM, 100 nM, 50 nM, ST:STUDY_SUMMARY 20 nM, or 0 nM folic acid followed by folate metabolomics. 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_Titration_2000nM_folate_1 Treatment:2000_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM991.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_2000nM_folate_2 Treatment:2000_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM992.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_2000nM_folate_3 Treatment:2000_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM993.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_100nM_folate_1 Treatment:100_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM994.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_100nM_folate_2 Treatment:100_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM995.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_100nM_folate_3 Treatment:100_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM996.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_50nM_folate_1 Treatment:50_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM997.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_50nM_folate_2 Treatment:50_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM998.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_50nM_folate_3 Treatment:50_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM999.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_20nM_folate_1 Treatment:20_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM1000.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_20nM_folate_2 Treatment:20_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM1001.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_20nM_folate_3 Treatment:20_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM1002.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_0nM_folate_1 Treatment:0_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM1003.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_0nM_folate_2 Treatment:0_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM1004.raw SUBJECT_SAMPLE_FACTORS K562 K562_Titration_0nM_folate_3 Treatment:0_nM_FA RAW_FILE_NAME=Copy of 20230222_QEF2_AM_C18_Folate_titration_AM1005.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 48hr in RPMI media containing 2000 nM, 100 nM, 50 nM, TR:TREATMENT_SUMMARY 20 nM, or 0 nM folic acid followed by folate metabolomics. #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 folate metabolite detection. Folate samples were resuspended SP:SAMPLEPREP_SUMMARY in reconstitution buffer (0.5% ascorbic acid, 1% K2HPO4 and 0.5% SP:SAMPLEPREP_SUMMARY 2-mercaptoethanol) containing rat serum (Sigma, R9759). The rat serum contains SP:SAMPLEPREP_SUMMARY the enzymes required to strip the polyglutamate tail from the measured folate. SP:SAMPLEPREP_SUMMARY Rat endogenous folate was removed from the rat serum by activated carbon SP:SAMPLEPREP_SUMMARY treatment (Sigma, C9157). Polyglutamate tail removal was carried out for 2 h SP:SAMPLEPREP_SUMMARY at 37 °C. After the polyglutamate tail removal, the pH was adjusted to 4 SP:SAMPLEPREP_SUMMARY using formic acid and samples were loaded on Bond Elute-pH columns (Agilent, SP:SAMPLEPREP_SUMMARY 14102062) for a pH-based clean-up. Columns were eluted with 300 μl elution SP:SAMPLEPREP_SUMMARY buffer (50% methanol, 0.25% NH4OAc, 0.5% 2-mercaptoethanol), followed by drying SP:SAMPLEPREP_SUMMARY the samples using a liquid nitrogen dryer manifold and resuspension in 25 μl SP:SAMPLEPREP_SUMMARY water. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 5 μl 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 The column oven and autosampler tray were held at 30 °C and 4 °C, CH:CHROMATOGRAPHY_SUMMARY respectively. The following conditions were used to achieve chromatographic CH:CHROMATOGRAPHY_SUMMARY separation: buffer A was 0.1% formic acid; buffer B was acetonitrile with 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid. The chromatographic gradient was run at a flow rate of 0.250 ml CH:CHROMATOGRAPHY_SUMMARY min−1 as follows: 0–5 min: gradient was held at 5% B; 5–10 min: linear CH:CHROMATOGRAPHY_SUMMARY gradient of 5% to 36% B; 10.1–14.0 min: linear gradient from 36–95% B; CH:CHROMATOGRAPHY_SUMMARY 14.1–18.0 min: gradient was returned 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 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0.250 ml min−1 as follows: 0–5 min: gradient was held at 5% B; CH:FLOW_GRADIENT 5–10 min: linear gradient of 5% to 36% B; 10.1–14.0 min: linear gradient CH:FLOW_GRADIENT from 36–95% B; 14.1–18.0 min: gradient was returned to 5% B. CH:FLOW_RATE 250 uL/min CH:COLUMN_TEMPERATURE 30 #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 The mass spectrometer was operated in full-scan, positive ionization mode using MS:MS_COMMENTS three narrow-range scans: 438–450 m/z; 452–462 m/z; and 470–478 m/z, with MS:MS_COMMENTS the resolution set at 70,000, the AGC target at 10e6, and the maximum injection MS:MS_COMMENTS time of 150 ms. HESI settings were: sheath gas flow rate: 40; Aux gas flow MS:MS_COMMENTS rate: 10; Sweep gas: 0; Spray voltage: 2.8 (neg) 3.5 (pos); Capillary MS:MS_COMMENTS temperature 300; S-lens RF level 50; Aux gas heater temp: 350. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples K562_Titration_2000nM_folate_1 K562_Titration_2000nM_folate_2 K562_Titration_2000nM_folate_3 K562_Titration_100nM_folate_1 K562_Titration_100nM_folate_2 K562_Titration_100nM_folate_3 K562_Titration_50nM_folate_1 K562_Titration_50nM_folate_2 K562_Titration_50nM_folate_3 K562_Titration_20nM_folate_1 K562_Titration_20nM_folate_2 K562_Titration_20nM_folate_3 K562_Titration_0nM_folate_1 K562_Titration_0nM_folate_2 K562_Titration_0nM_folate_3 Factors Treatment:2000_nM_FA Treatment:2000_nM_FA Treatment:2000_nM_FA Treatment:100_nM_FA Treatment:100_nM_FA Treatment:100_nM_FA Treatment:50_nM_FA Treatment:50_nM_FA Treatment:50_nM_FA Treatment:20_nM_FA Treatment:20_nM_FA Treatment:20_nM_FA Treatment:0_nM_FA Treatment:0_nM_FA Treatment:0_nM_FA 5,10-methylenetetrahydrofolate 460879.6844 551216.0663 709526.196 48284.17254 31131.22155 38343.95336 35614.57635 31355.34678 41068.74338 35372.99792 42761.93389 41008.53853 17933.3537 18585.58252 37225.7861 5-methyltetrahydrofolate 2777081.557 3466600.95 3475538.459 793881.7261 664628.0992 649318.9832 643026.7874 622084.9707 493783.2339 697671.6118 800302.223 711111.9619 616206.8187 595797.409 709636.5853 5-formyltetrahydrofolate 32084.60687 42102.88358 45620.12569 3390.370441 2777.939018 1157.567354 5384.80266 1054.646232 1278.462597 2443.176682 2027.500357 2555.808991 3037.115676 2784.280402 365.1333086 10-formyltetrahydrofolate 19856.65369 26526.51538 23058.23402 15435.72093 10699.90392 7824.745887 22304.63838 13831.41222 10386.70822 8835.429527 12759.92596 8806.405751 18393.23241 10591.46622 12451.04582 Tetrahydrofolate 21567.95935 47100.38224 24683.39377 6298.672586 5552.877293 4683.30908 5957.024607 7664.042737 7298.335307 5411.407337 10173.19722 5803.065061 17345.09218 8445.171243 5940.986102 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Formula Exact mass Sub class 5,10-methylenetetrahydrofolate 5,10-Methylene-THF C20H23N7O6 457.1710 Tetrahydrofolic acids 5-methyltetrahydrofolate 5-Methyltetrahydrofolic acid C20H25N7O6 459.1866 Pterins 5-formyltetrahydrofolate N5-Formyl-THF C20H23N7O7 473.1659 Tetrahydrofolic acids 10-formyltetrahydrofolate N10-Formyl-THF C20H23N7O7 473.1659 Tetrahydrofolic acids Tetrahydrofolate THF C19H23N7O6 445.1710 Tetrahydrofolic acids METABOLITES_END #END