#METABOLOMICS WORKBENCH amaynard_20231001_210302 DATATRACK_ID:4360 STUDY_ID:ST002900 ANALYSIS_ID:AN004759 PROJECT_ID:PR001773
VERSION             	1
CREATED_ON             	October 1, 2023, 9:09 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 metabolite levels in erythroid progenitor cells following SHIN1 and
ST:STUDY_TITLE                   	inosine treatment
ST:STUDY_SUMMARY                 	Culture of erythroid progenitor cells cultured in SFEM II media supplemented
ST:STUDY_SUMMARY                 	with SHIN1 and/or inosine for 48 hours followed up by metabolomics targeting
ST:STUDY_SUMMARY                 	folate 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                    	4
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:CELL_BIOSOURCE_OR_SUPPLIER    	Lineage depleted erythroid progenitor cells isolated from E14.5 mice.
#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           	Erythroid_Progenitor	EP_Vehicle_folate_1	Treatment:Vehicle	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM916.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_Vehicle_folate_2	Treatment:Vehicle	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM917.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_Vehicle_folate_3	Treatment:Vehicle	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM918.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_SHIN1_folate_1	Treatment:SHIN1	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM919.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_SHIN1_folate_2	Treatment:SHIN1	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM920.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_SHIN1_folate_3	Treatment:SHIN1	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM921.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_Inosine_folate_1	Treatment:Inosine	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM922.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_Inosine_folate_2	Treatment:Inosine	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM923.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_Inosine_folate_3	Treatment:Inosine	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM924.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_SHIN1_Inosine_folate_1	Treatment:SHIN1_Inosine	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM925.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_SHIN1_Inosine_folate_2	Treatment:SHIN1_Inosine	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM926.raw
SUBJECT_SAMPLE_FACTORS           	Erythroid_Progenitor	EP_SHIN1_Inosine_folate_3	Treatment:SHIN1_Inosine	RAW_FILE_NAME=Copy of 20220715_AM_ErythProg3_Folates_AM927.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             	Erythroid progenitor cells were cultured for 48hr in SFEM II media containing
TR:TREATMENT_SUMMARY             	vehicle, SHIN1 (1.25 uM), inosine (100 uM), or SHIN1+inosine.
#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 2.1mm,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	EP_Vehicle_folate_1	EP_Vehicle_folate_2	EP_Vehicle_folate_3	EP_SHIN1_folate_1	EP_SHIN1_folate_2	EP_SHIN1_folate_3	EP_Inosine_folate_1	EP_Inosine_folate_2	EP_Inosine_folate_3	EP_SHIN1_Inosine_folate_1	EP_SHIN1_Inosine_folate_2	EP_SHIN1_Inosine_folate_3
Factors	Treatment:Vehicle	Treatment:Vehicle	Treatment:Vehicle	Treatment:SHIN1	Treatment:SHIN1	Treatment:SHIN1	Treatment:Inosine	Treatment:Inosine	Treatment:Inosine	Treatment:SHIN1_Inosine	Treatment:SHIN1_Inosine	Treatment:SHIN1_Inosine
5-methyltetrahydrofolate	161171.709	137400.074	140644.706	103179.794	81368.2934	91220.7011	151373.917	121432.944	134154.499	93862.3027	76055.8534	93658.032
5,10-methylenetetrahydrofolate	360115.416	325878.597	350988.259	153690.327	140368.139	135719.96	403155.701	308782.449	242878.861	232081.344	254667.65	268475.869
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Standardized name	Formula	Exact mass	Sub class
5-methyltetrahydrofolate	5-Methyltetrahydrofolic acid	C20H25N7O6	459.1866	Pterins
5,10-methylenetetrahydrofolate	-	-	-	-
METABOLITES_END
#END