#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