#METABOLOMICS WORKBENCH amaynard_20230928_181920 DATATRACK_ID:4339 STUDY_ID:ST002892 ANALYSIS_ID:AN004751 PROJECT_ID:PR001773 VERSION 1 CREATED_ON October 2, 2023, 8:26 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 levels in K562 cells following folate depletion ST:STUDY_SUMMARY Culture of K562 cells in RPMI media containing 100 nM folic acid for up to 8 ST:STUDY_SUMMARY days followed my metabolomics targeting folate metabolites. All samples were ST:STUDY_SUMMARY harvested at the same time and the study was set up as a reverse time-course. ST:STUDY_SUMMARY Day 0 samples were cultured in 2,000 nM folate for the full 8 days. 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_100nM_Timecourse_Day0_Folate_1 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM591.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day0_Folate_2 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM592.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day0_Folate_3 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM593.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day0_Folate_4 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM594.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day1_Folate_1 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM595.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day1_Folate_2 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM596.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day1_Folate_3 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM597.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day1_Folate_4 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM598.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day2_Folate_1 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM599.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day2_Folate_2 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM600.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day2_Folate_3 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM601.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day2_Folate_4 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM602.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day4_Folate_1 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM603.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day4_Folate_2 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM604.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day4_Folate_3 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM605.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day4_Folate_4 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM606.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day6_Folate_1 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM607.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day6_Folate_2 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM608.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day6_Folate_3 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM609.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day6_Folate_4 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM610.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day8_Folate_1 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM611.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day8_Folate_2 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM612.raw SUBJECT_SAMPLE_FACTORS K562 K562_100nM_Timecourse_Day8_Folate_3 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20211130_Adam_K562_TC_Folate_AM613.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 K562 cells were cultured for up to 8 days in either 2,000 nM folic acid or 100 TR:TREATMENT_SUMMARY nM folic acid containing RPMI media. Experiment was a reverse time-course. TR:TREATMENT_SUMMARY Samples were cultured for the indicated length of time in 100 nM folic acid and TR:TREATMENT_SUMMARY harvested on the same date. Day 0 samples were cultured in 2,000 nM folic acid TR:TREATMENT_SUMMARY for the full 8 day experiment #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 Arbitrary Units MS_METABOLITE_DATA_START Samples K562_100nM_Timecourse_Day0_Folate_1 K562_100nM_Timecourse_Day0_Folate_2 K562_100nM_Timecourse_Day0_Folate_3 K562_100nM_Timecourse_Day0_Folate_4 K562_100nM_Timecourse_Day1_Folate_1 K562_100nM_Timecourse_Day1_Folate_2 K562_100nM_Timecourse_Day1_Folate_3 K562_100nM_Timecourse_Day1_Folate_4 K562_100nM_Timecourse_Day2_Folate_1 K562_100nM_Timecourse_Day2_Folate_2 K562_100nM_Timecourse_Day2_Folate_3 K562_100nM_Timecourse_Day2_Folate_4 K562_100nM_Timecourse_Day4_Folate_1 K562_100nM_Timecourse_Day4_Folate_2 K562_100nM_Timecourse_Day4_Folate_3 K562_100nM_Timecourse_Day4_Folate_4 K562_100nM_Timecourse_Day6_Folate_1 K562_100nM_Timecourse_Day6_Folate_2 K562_100nM_Timecourse_Day6_Folate_3 K562_100nM_Timecourse_Day6_Folate_4 K562_100nM_Timecourse_Day8_Folate_1 K562_100nM_Timecourse_Day8_Folate_2 K562_100nM_Timecourse_Day8_Folate_3 Factors 100nM_Folic_Acid_Growth:Day0 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:Day6 100nM_Folic_Acid_Growth:Day8 100nM_Folic_Acid_Growth:Day8 100nM_Folic_Acid_Growth:Day8 5,10-Methylenetetrahydrofolate 90107.64217 82790.79124 110023.1188 188542.8353 86129.17182 205292.1885 47926.19904 28353.69465 27600.48078 34811.56416 31315.71011 68633.86958 24854.02712 36405.83868 173408.149 19941.01383 29255.6949 41601.52081 67376.04889 32147.72662 36169.9172 28536.88366 63411.02661 Folic Acid 742139.1736 859811.0433 878147.3172 911221.066 108751.9155 255701.5607 96759.78345 75850.19844 109395.983 120204.2362 116794.4772 154412.2259 86061.31882 104839.323 270852.2535 85754.842 118292.1775 124181.8066 126383.6301 134083.622 118814.2147 119922.3329 160343.6091 5-methyltetrahydrofolate 928100.2985 954688.0815 1226331.188 1404617.031 552192.7623 1408679.155 457393.5517 417083.6811 224884.5465 215422.243 304345.2492 356587.2379 163320.2897 195050.2765 834202.6318 136884.4686 180425.5321 267382.1686 365604.7184 157208.6302 150960.6169 173876.0236 236065.5345 tetrahydrofolate 41798.64209 46981.61984 60553.55877 106663.4481 36905.45694 127561.4832 37595.54893 22852.70507 18998.9814 19982.6599 30642.99522 34211.63345 17948.52998 25662.69992 107023.4577 9004.44102 20488.99466 24995.09831 49578.48247 19434.39951 19612.31829 17787.00148 25401.60128 5-formyltetrahydrofolate 91975.97051 127792.5144 180650.4822 421804.2675 229964.56 1274044.488 150210.6502 34628.86819 71103.83023 64186.1269 146412.9491 170629.8228 116614.542 156736.7884 865435.393 72121.53158 162106.6758 223898.0367 402593.751 107110.7224 105824.0089 124704.1297 195119.8488 10-formyltetrahydrofolate 14818.17363 17374.38713 16710.40786 20134.34932 15003.28747 31504.19885 9286.931226 9036.376819 8812.651898 7899.263499 11877.10229 12136.42615 9309.455325 5877.715547 19455.87405 7403.363518 7814.018367 6562.24426 10994.06809 4123.690263 4774.447611 5805.944038 7267.169383 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Formula Exact mass Sub class 5,10-Methylenetetrahydrofolate - - - - Folic Acid Folic acid C19H19N7O6 441.1397 Folic acids 5-methyltetrahydrofolate 5-Methyltetrahydrofolic acid C20H25N7O6 459.1866 Pterins tetrahydrofolate 6R-THF C19H23N7O6 445.1710 Tetrahydrofolic acids 5-formyltetrahydrofolate - - - - 10-formyltetrahydrofolate N10-Formyl-THF C20H23N7O7 473.1659 Tetrahydrofolic acids METABOLITES_END #END