Summary of Study ST002900

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	ST002900
Study Title	Folate metabolite levels in erythroid progenitor cells following SHIN1 and inosine treatment
Study Summary	Culture of erythroid progenitor cells cultured in SFEM II media supplemented with SHIN1 and/or inosine for 48 hours followed up by metabolomics targeting folate 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	2023-10-01
Num Groups	4
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:	SU003013
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090
Cell Biosource Or Supplier:	Lineage depleted erythroid progenitor cells isolated from E14.5 mice.

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA315602	EP_Inosine_folate_3	Inosine
SA315603	EP_Inosine_folate_2	Inosine
SA315604	EP_Inosine_folate_1	Inosine
SA315605	EP_SHIN1_folate_2	SHIN1
SA315606	EP_SHIN1_folate_1	SHIN1
SA315607	EP_SHIN1_folate_3	SHIN1
SA315608	EP_SHIN1_Inosine_folate_1	SHIN1_Inosine
SA315609	EP_SHIN1_Inosine_folate_3	SHIN1_Inosine
SA315610	EP_SHIN1_Inosine_folate_2	SHIN1_Inosine
SA315611	EP_Vehicle_folate_2	Vehicle
SA315612	EP_Vehicle_folate_3	Vehicle
SA315613	EP_Vehicle_folate_1	Vehicle

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO003006
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:	TR003022
Treatment Summary:	Erythroid progenitor cells were cultured for 48hr in SFEM II media containing vehicle, SHIN1 (1.25 uM), inosine (100 uM), or SHIN1+inosine.

Sample Preparation:

Sampleprep ID:	SP003019
Sampleprep Summary:	Pellet was resuspended in extraction buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000 g to pellet cell debris. The supernatant was divided into two tubes and dried on ice using a liquid nitrogen dryer. One tube of dried sample was used for folate metabolite detection. Folate samples were resuspended in reconstitution buffer (0.5% ascorbic acid, 1% K2HPO4 and 0.5% 2-mercaptoethanol) containing rat serum (Sigma, R9759). The rat serum contains the enzymes required to strip the polyglutamate tail from the measured folate. Rat endogenous folate was removed from the rat serum by activated carbon treatment (Sigma, C9157). Polyglutamate tail removal was carried out for 2 h at 37 °C. After the polyglutamate tail removal, the pH was adjusted to 4 using formic acid and samples were loaded on Bond Elute-pH columns (Agilent, 14102062) for a pH-based clean-up. Columns were eluted with 300 μl elution buffer (50% methanol, 0.25% NH4OAc, 0.5% 2-mercaptoethanol), followed by drying the samples using a liquid nitrogen dryer manifold and resuspension in 25 μl water.

Sampleprep ID:

SP003019

Sampleprep Summary:

Pellet was resuspended in extraction buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000 g to pellet cell debris. The supernatant was divided into two tubes and dried on ice using a liquid nitrogen dryer. One tube of dried sample was used for folate metabolite detection. Folate samples were resuspended in reconstitution buffer (0.5% ascorbic acid, 1% K2HPO4 and 0.5% 2-mercaptoethanol) containing rat serum (Sigma, R9759). The rat serum contains the enzymes required to strip the polyglutamate tail from the measured folate. Rat endogenous folate was removed from the rat serum by activated carbon treatment (Sigma, C9157). Polyglutamate tail removal was carried out for 2 h at 37 °C. After the polyglutamate tail removal, the pH was adjusted to 4 using formic acid and samples were loaded on Bond Elute-pH columns (Agilent, 14102062) for a pH-based clean-up. Columns were eluted with 300 μl elution buffer (50% methanol, 0.25% NH4OAc, 0.5% 2-mercaptoethanol), followed by drying the samples using a liquid nitrogen dryer manifold and resuspension in 25 μl water.

Combined analysis:

Analysis ID	AN004759
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	peak area

Chromatography:

Chromatography ID:	CH003591
Chromatography Summary:	5 μl 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). The column oven and autosampler tray were held at 30 °C and 4 °C, respectively. The following conditions were used to achieve chromatographic separation: buffer A was 0.1% formic acid; buffer B was acetonitrile with 0.1% formic acid. The chromatographic gradient was run at a flow rate of 0.250 ml min−1 as follows: 0–5 min: gradient was held at 5% B; 5–10 min: linear gradient of 5% to 36% B; 10.1–14.0 min: linear gradient from 36–95% B; 14.1–18.0 min: gradient was returned to 5% B.
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:	30
Flow Gradient:	0.250 ml min−1 as follows: 0–5 min: gradient was held at 5% B; 5–10 min: linear gradient of 5% to 36% B; 10.1–14.0 min: linear gradient from 36–95% B; 14.1–18.0 min: gradient was returned to 5% B.
Flow Rate:	250 uL/min
Solvent A:	0.1% formic acid
Solvent B:	acetonitrile with 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004505
Analysis ID:	AN004759
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 three narrow-range scans: 438–450 m/z; 452–462 m/z; and 470–478 m/z, with the resolution set at 70,000, the AGC target at 10e6, and the maximum injection time of 150 ms. HESI settings were: sheath gas flow rate: 40; Aux gas flow rate: 10; Sweep gas: 0; Spray voltage: 2.8 (neg) 3.5 (pos); Capillary temperature 300; S-lens RF level 50; Aux gas heater temp: 350.
Ion Mode:	UNSPECIFIED