Summary of Study ST003023

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001773. The data can be accessed directly via it's Project DOI: 10.21228/M8J420 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003023
Study Title	Porphyrin metabolite levels in K562 cells following 6 day culture in 2,000 nM or 100 nM folic acid
Study Summary	Culture of K562 cells in RPMI media containing 2000 nM or 100 nM folic acid for 6 days. These samples were followed up by metabolomics targeting porphyrin metabolites.
Institute	Boston Children's Hospital, Harvard Medical School
Department	pathology
Laboratory	Kanarek Lab
Last Name	Kanarek
First Name	Naama
Address	Enders 1116.2, 300 Longwood Ave, Boston, MA 02115
Email	naama.kanarek@childrens.harvard.edu
Phone	6173557433
Submit Date	2023-12-19
Num Groups	5
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-02-13
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001773
Project DOI:	doi: 10.21228/M8J420
Project Title:	Folate depletion induces erythroid differentiation through perturbation of de novo purine synthesis
Project Summary:	Folate, an essential vitamin, is a one-carbon acceptor and donor in key metabolic reactions. Erythroid cells harbor a unique sensitivity to folate deprivation, as revealed by the primary pathological manifestation of nutritional folate deprivation: megaloblastic anemia. To study this metabolic sensitivity, we applied mild folate depletion to human and mouse erythroid cell lines, and primary murine erythroid progenitors. We show that folate depletion induces early blockade of purine synthesis and accumulation of the purine synthesis intermediate and signaling molecule, AICAR, followed by enhanced heme metabolism, hemoglobin synthesis, and erythroid differentiation. This is phenocopied by inhibition of folate metabolism using the SHMT1/2 inhibitor - SHIN1, and by AICAR supplementation. Mechanistically, the metabolically-driven differentiation is independent of nucleotide sensing through mTORC1 and AMPK, and is instead mediated by protein kinase C (PKC). Our findings suggest that folate deprivation-induced premature differentiation of erythroid progenitor cells is a molecular etiology to folate-deficiency induced anemia.
Institute:	Boston Children's Hospital, Harvard Medical School
Department:	pathology
Laboratory:	Kanarek Lab
Last Name:	Kanarek
First Name:	Naama
Address:	Enders 1116.2, 300 Longwood Ave, Boston, MA 02115
Email:	naama.kanarek@childrens.harvard.edu
Phone:	(617) 355-7433

Subject:

Subject ID:	SU003137
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Strain Details:	K-562

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Folic_Acid_Concentration
SA327717	K562_100nM_Porpyrin_3	100_nM
SA327718	K562_100nM_Porpyrin_4	100_nM
SA327719	K562_100nM_Porpyrin_2	100_nM
SA327720	K562_100nM_Porpyrin_1	100_nM
SA327721	K562_2000nM_Porphyrin_2	2000_nM
SA327722	K562_2000nM_Porphyrin_3	2000_nM
SA327723	K562_2000nM_Porphyrin_4	2000_nM
SA327724	K562_2000nM_Porphyrin_1	2000_nM

Showing results 1 to 8 of 8

Showing results 1 to 8 of 8

Collection:

Collection ID:	CO003130
Collection Summary:	One million cells from culture were collected via centrifugation for 20 seconds at 18,000xG, washed with 0.9% NaCl, and collected via centrifugation for 20 seconds at 18,000xG
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR003146
Treatment Summary:	Culture of K562 cells for 6 days in RPMI media containing 2000 nM or 100 nM folic acid for 6 days. These samples were followed up by metabolomics targeting porphyrin metabolites.

Sample Preparation:

Sampleprep ID:	SP003143
Sampleprep Summary:	One million cells from culture were collected via centrifugation, washed with 0.9% NaCl, and resuspended in 150 µl of porphyrin extraction buffer (1:4 ratio of 1.7 M HCl:ACN, 1µM deuteroporphyrin IX (Frontier Scientific, D510-9)) and 0.5 µM isotopically labeled amino acids (Cambridge Isotopes, MSK-A2-1.2)). Samples were vigorously shaken for 20 min at 16ºC in a thermomixer (Eppendorf), sonicated for 10 cycles at 4ºC with 30 sec on and 30 sec off, then incubated at 4ºC for 10 min. Following incubation on ice, samples were centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was collected and 40.5 µl super-saturated MgSO4 and 12 µl 5 M NaCl were added. Samples were vortexed for 30 sec and further shaken for 10 min at 16 ºC in a thermomixer. Finally, a 10 min 10,000 rpm centrifugation was used to separate the organic layer (upper) from the aqueous layer (lower). The upper organic layer was collected.

Sampleprep ID:

SP003143

Sampleprep Summary:

One million cells from culture were collected via centrifugation, washed with 0.9% NaCl, and resuspended in 150 µl of porphyrin extraction buffer (1:4 ratio of 1.7 M HCl:ACN, 1µM deuteroporphyrin IX (Frontier Scientific, D510-9)) and 0.5 µM isotopically labeled amino acids (Cambridge Isotopes, MSK-A2-1.2)). Samples were vigorously shaken for 20 min at 16ºC in a thermomixer (Eppendorf), sonicated for 10 cycles at 4ºC with 30 sec on and 30 sec off, then incubated at 4ºC for 10 min. Following incubation on ice, samples were centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was collected and 40.5 µl super-saturated MgSO4 and 12 µl 5 M NaCl were added. Samples were vortexed for 30 sec and further shaken for 10 min at 16 ºC in a thermomixer. Finally, a 10 min 10,000 rpm centrifugation was used to separate the organic layer (upper) from the aqueous layer (lower). The upper organic layer was collected.

Combined analysis:

Analysis ID	AN004957
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Phenomenex Kinetex C18 (150 x 3mm,2.6um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE
Units	Arbitrary Units

Chromatography:

Chromatography ID:	CH003741
Chromatography Summary:	Sample (5 uL) was injected onto a 2.6 μm, 150 x 3 mm C18 column (Phenomenex, 00F-4462-Y0) equipped with a 3.0 mm safe-guard column (Phenomenex, AJ0-8775). Column compartment was heated to 45 ºC. Porphyrins were separated with a chromatographic gradient at a flow rate of 0.800 ml min−1 as follows: 0–2 min: 5% B; 2-19min: linear gradient from 5% to 95% B; 19-21min: 95% B; 21.1-23min: return to 5% B.
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 3mm,2.6um)
Column Temperature:	45
Flow Gradient:	0–2 min: 5% B; 2-19min: linear gradient from 5% to 95% B; 19-21min: 95% B; 21.1-23min: return to 5% B.
Flow Rate:	0.8 mL/min
Solvent A:	95% water/5% acetonitrile; 0.1% formic acid
Solvent B:	5% water/95% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004697
Analysis ID:	AN004957
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The mass spectrometer was operated in full-scan, positive ionization mode using a narrow-range scan: 450-700m/z, with an additional tSIM scan for hemin (616.1767 m/z), CoproP (655.2762 m/z), and PPIX (563.2653 m/z). Metabolites were relatively quantified while referencing an in-house library of chemical standards and using TraceFinder 4.1 (Thermo Fisher Scientific, Waltham, MA, USA), with a 5 ppm mass tolerance.
Ion Mode:	POSITIVE