#METABOLOMICS WORKBENCH amaynard_20231001_171634 DATATRACK_ID:4353 STUDY_ID:ST002894 ANALYSIS_ID:AN004753 PROJECT_ID:PR001773 VERSION 1 CREATED_ON October 1, 2023, 5:29 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 MEL cells following folate depletion ST:STUDY_SUMMARY Culture of MEL cells in RPMI media containing 100 nM folic acid for up to 8 days ST:STUDY_SUMMARY 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 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_folate_1 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM665.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day0_folate_2 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM666.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day0_folate_3 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM667.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day0_folate_4 100nM_Folic_Acid_Growth:Day0 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM668.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day1_folate_1 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM669.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day1_folate_2 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM670.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day1_folate_3 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM671.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day1_folate_4 100nM_Folic_Acid_Growth:Day1 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM672.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day2_folate_1 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM673.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day2_folate_2 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM674.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day2_folate_3 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM675.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day2_folate_4 100nM_Folic_Acid_Growth:Day2 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM676.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day4_folate_1 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM677.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day4_folate_2 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM678.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day4_folate_3 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM679.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day4_folate_4 100nM_Folic_Acid_Growth:Day4 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM680.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day6_folate_1 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM681.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day6_folate_2 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM682.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day6_folate_3 100nM_Folic_Acid_Growth:Day6 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM683.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day8_folate_1 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM684.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day8_folate_2 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM685.raw SUBJECT_SAMPLE_FACTORS MEL MEL_100nM_Timecourse_Day8_folate_3 100nM_Folic_Acid_Growth:Day8 RAW_FILE_NAME=Copy of 20220325_AM_MEL_Timecourse_folates_AM686.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 up to 8 days in either 2,000 nM folic acid or 100 nM TR:TREATMENT_SUMMARY folic acid containing RPMI media. Experiment was a reverse time-course. Samples TR:TREATMENT_SUMMARY 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 0.1% formic acid CH:SOLVENT_B acetonitrile with 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 MEL_100nM_Timecourse_Day0_folate_1 MEL_100nM_Timecourse_Day0_folate_2 MEL_100nM_Timecourse_Day0_folate_3 MEL_100nM_Timecourse_Day0_folate_4 MEL_100nM_Timecourse_Day1_folate_1 MEL_100nM_Timecourse_Day1_folate_2 MEL_100nM_Timecourse_Day1_folate_3 MEL_100nM_Timecourse_Day1_folate_4 MEL_100nM_Timecourse_Day2_folate_1 MEL_100nM_Timecourse_Day2_folate_2 MEL_100nM_Timecourse_Day2_folate_3 MEL_100nM_Timecourse_Day2_folate_4 MEL_100nM_Timecourse_Day4_folate_1 MEL_100nM_Timecourse_Day4_folate_2 MEL_100nM_Timecourse_Day4_folate_3 MEL_100nM_Timecourse_Day4_folate_4 MEL_100nM_Timecourse_Day6_folate_1 MEL_100nM_Timecourse_Day6_folate_2 MEL_100nM_Timecourse_Day6_folate_3 MEL_100nM_Timecourse_Day8_folate_1 MEL_100nM_Timecourse_Day8_folate_2 MEL_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:Day8 100nM_Folic_Acid_Growth:Day8 100nM_Folic_Acid_Growth:Day8 Folic Acid 290569.8984 508996.5727 537439.715 420610.3609 15259.77632 79218.70714 105363.2119 93670.01663 50190.54651 27553.92111 37239.11813 38465.38428 42377.83886 21159.35375 50361.77664 28095.77724 41728.64793 38365.62971 60443.83538 22116.2439 22762.89944 44488.30521 Tetrahydrofolate 649802.0803 736379.8822 606554.4786 566417.9614 153285.7881 216384.1582 187965.1843 155454.2368 42314.45732 35156.86496 33138.71961 29605.13178 22184.87085 26987.64031 1752.066097 12375.12881 11275.51506 4364.862921 17676.38043 9633.455933 4172.641616 11019.43276 10-Formyltetrahydrofolate 104128.1786 115658.3505 120553.6195 116742.2375 16587.904 29365.72764 26817.31106 23597.03936 19620.47143 14597.51839 16952.37209 11780.5674 8465.233096 3338.699201 11600.35134 5728.79062 5564.239826 6067.426879 3095.042591 2893.100578 4325.874218 0 5-Formyltetrahydrofolate 105135.3068 116325.7558 122766.6156 118557.5162 16587.904 31561.77667 27346.13736 23597.03936 18846.25704 14811.14061 17473.93109 11780.09224 8850.305995 3347.254484 11603.05098 5728.79062 4948.791631 8494.991894 3128.562908 2949.999682 4392.667916 0 5-methyltetrahydrofolate 552214.2237 767121.6554 659342.2267 762013.1968 109202.5603 190158.2283 164979.0417 151219.4448 41594.15447 41252.02782 39974.50275 33711.43854 19223.91508 29496.47706 21440.53766 11568.15749 15222.36543 9080.341108 14590.27659 11769.36075 15382.19579 5140.828875 5,10-methyltetrahydrofolate 913221.1339 1131007.113 1128228.41 1026089.842 142325.3117 249069.672 208707.5056 194834.0135 84732.84722 66520.62128 75867.59932 52383.20784 66372.69391 90399.39811 112769.3451 99448.04086 87561.68417 89403.92133 77494.50323 54423.99264 49138.55214 43308.49442 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Formula Exact mass Sub class Folic Acid Folic acid C19H19N7O6 441.1397 Folic acids Tetrahydrofolate 6R-THF C19H23N7O6 445.1710 Tetrahydrofolic acids 10-Formyltetrahydrofolate N10-Formyl-THF C20H23N7O7 473.1659 Tetrahydrofolic acids 5-Formyltetrahydrofolate - - - - 5-methyltetrahydrofolate 5-Methyltetrahydrofolic acid C20H25N7O6 459.1866 Pterins 5,10-methyltetrahydrofolate - - - - METABOLITES_END #END