Summary of Study ST002899

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.

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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.

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Study IDST002899
Study TitlePolar metabolite levels in erythroid progenitor cells following SHIN1 and inosine treatment
Study SummaryCulture of erythroid progenitor cells cultured in SFEM II media supplemented with SHIN1 and/or inosine for 48 hours followed up by metabolomics targeting polar metabolites.
Institute
Boston Children's Hospital, Harvard Medical School
Departmentpathology
LaboratoryKanarek Lab
Last NameKanarek
First NameNaama
AddressEnders 1116.2, 300 Longwood Ave, Boston, MA 02115
Emailnaama.kanarek@childrens.harvard.edu
Phone6173557433
Submit Date2023-10-01
Num Groups4
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-02-13
Release Version1
Naama Kanarek Naama Kanarek
https://dx.doi.org/10.21228/M8J420
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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:SU003012
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.
Species Group:Mammals

Factors:

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

mb_sample_id local_sample_id Treatment
SA315590EP_Inosine_HILIC_3Inosine
SA315591EP_Inosine_HILIC_2Inosine
SA315592EP_Inosine_HILIC_1Inosine
SA315593EP_SHIN1_HILIC_2SHIN1
SA315594EP_SHIN1_HILIC_1SHIN1
SA315595EP_SHIN1_HILIC_3SHIN1
SA315596EP_SHIN1_Inosine_HILIC_1SHIN1_Inosine
SA315597EP_SHIN1_Inosine_HILIC_3SHIN1_Inosine
SA315598EP_SHIN1_Inosine_HILIC_2SHIN1_Inosine
SA315599EP_Vehicle_HILIC_2Vehicle
SA315600EP_Vehicle_HILIC_3Vehicle
SA315601EP_Vehicle_HILIC_1Vehicle
Showing results 1 to 12 of 12

Collection:

Collection ID:CO003005
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:TR003021
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:SP003018
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 polar metabolite detection. Dried samples were resuspended in 25 μL water

Combined analysis:

Analysis ID AN004758
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column EMD Millipore ZIC-HILIC (100 x 2.1 mm,3.5 um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units peak area

Chromatography:

Chromatography ID:CH003590
Chromatography Summary:Sample was injected into a ZIC-pHILIC 150 x 2.1 mm (5 μm particle size) column (EMD Millipore). operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separation was achieved using the following conditions: buffer A was acetonitrile; buffer B was 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water; resulting pH is around 9 without pH adjustment. Gradient conditions we used were: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively.
Instrument Name:Thermo Vanquish
Column Name:EMD Millipore ZIC-HILIC (100 x 2.1 mm,3.5 um)
Column Temperature:25
Flow Gradient:linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B at 150 mL/min flow rate
Flow Rate:150 mL/min
Solvent A:100% acetonitrile
Solvent B:100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide
Chromatography Type:HILIC

MS:

MS ID:MS004504
Analysis ID:AN004758
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Polar 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:UNSPECIFIED
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