Summary of Study ST002905

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	ST002905
Study Title	Porphyrin analysis of K562 cells following C13-Serine and C13-Glycine tracing during folate depletion.
Study Summary	Culture of K562 cells for 7 days in RPMI media containing 2000 nM folic acid or 100 nM folic acid. At day 8, media was changed to 2000 nM or 100 nM folic acid with unlabeled serine and glycine, or 2-C13-Serine and 2-C13-Glycine at RPMI levels. Amino acid tracing was performed for 24 hours. 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	2023-10-02
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:	SU003018
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	Treatment
SA315921	K562_100nM_FA_C13_Glycine_porphyrin_4	100nM_FA_C13_Glycine
SA315922	K562_100nM_FA_C13_Glycine_porphyrin_1	100nM_FA_C13_Glycine
SA315923	K562_100nM_FA_C13_Glycine_porphyrin_2	100nM_FA_C13_Glycine
SA315924	K562_100nM_FA_C13_Glycine_porphyrin_3	100nM_FA_C13_Glycine
SA315925	K562_100nM_FA_C13_Serine_porphyrin_1	100nM_FA_C13_Serine
SA315926	K562_100nM_FA_C13_Serine_porphyrin_2	100nM_FA_C13_Serine
SA315927	K562_100nM_FA_C13_Serine_porphyrin_4	100nM_FA_C13_Serine
SA315928	K562_100nM_FA_C13_Serine_porphyrin_3	100nM_FA_C13_Serine
SA315929	K562_100nM_FA_Unlabeled_porphyrin_3	100nM_FA_Unlabeled
SA315930	K562_100nM_FA_Unlabeled_porphyrin_1	100nM_FA_Unlabeled
SA315931	K562_100nM_FA_Unlabeled_porphyrin_2	100nM_FA_Unlabeled
SA315932	K562_100nM_FA_Unlabeled_porphyrin_4	100nM_FA_Unlabeled
SA315933	K562_2000nM_FA_C13_Glycine_porphyrin_1	2000nM_FA_C13_Glycine
SA315934	K562_2000nM_FA_C13_Glycine_porphyrin_2	2000nM_FA_C13_Glycine
SA315935	K562_2000nM_FA_C13_Glycine_porphyrin_3	2000nM_FA_C13_Glycine
SA315936	K562_2000nM_FA_C13_Glycine_porphyrin_4	2000nM_FA_C13_Glycine
SA315937	K562_2000nM_FA_C13_Serine_porphyrin_1	2000nM_FA_C13_Serine
SA315938	K562_2000nM_FA_C13_Serine_porphyrin_4	2000nM_FA_C13_Serine
SA315939	K562_2000nM_FA_C13_Serine_porphyrin_2	2000nM_FA_C13_Serine
SA315940	K562_2000nM_FA_C13_Serine_porphyrin_3	2000nM_FA_C13_Serine
SA315941	K562_2000nM_FA_Unlabeled_porphyrin_2	2000nM_FA_Unlabeled
SA315942	K562_2000nM_FA_Unlabeled_porphyrin_3	2000nM_FA_Unlabeled
SA315943	K562_2000nM_FA_Unlabeled_porphyrin_1	2000nM_FA_Unlabeled
SA315944	K562_2000nM_FA_Unlabeled_porphyrin_4	2000nM_FA_Unlabeled

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO003011
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:	TR003027
Treatment Summary:	Culture of K562 cells for 7 days in RPMI media containing 2000 nM folic acid or 100 nM folic acid. At day 8, media was changed to 2000 nM or 100 nM folic acid with unlabeled serine and glycine, or 2-C13-Serine and 2-C13-Glycine at RPMI levels. Amino acid tracing was performed for 24 hours. The level of serine and glycine in all conditions was 30 and 10 mg/L, respectively.

Sample Preparation:

Sampleprep ID:	SP003024
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:

SP003024

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	AN004768
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	peak area

Chromatography:

Chromatography ID:	CH003600
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:	linear gradient from 5% to 95%
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:	MS004514
Analysis ID:	AN004768
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