Summary of Study ST002912

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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST002912
Study TitlePolar metabolite levels in MEL cells following folate depletion
Study SummaryCulture of MEL cells in RPMI media containing 100 nM folic acid for up to 8 days followed my metabolomics targeting polar metabolites. All samples were harvested at the same time and the study was set up as a reverse time-course. Day 0 samples were cultured in 2,000 nM folate for the full 8 days.
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 Groups6
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

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:SU003025
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Cell Strain Details:MEL

Factors:

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

mb_sample_id local_sample_id 100nM_Folic_Acid_Growth
SA316320MEL_100nM_Timecourse_Day0_HILIC_1Day0
SA316321MEL_100nM_Timecourse_Day0_HILIC_4Day0
SA316322MEL_100nM_Timecourse_Day0_HILIC_3Day0
SA316323MEL_100nM_Timecourse_Day0_HILIC_2Day0
SA316324MEL_100nM_Timecourse_Day1_HILIC_4Day1
SA316325MEL_100nM_Timecourse_Day1_HILIC_1Day1
SA316326MEL_100nM_Timecourse_Day1_HILIC_3Day1
SA316327MEL_100nM_Timecourse_Day1_HILIC_2Day1
SA316328MEL_100nM_Timecourse_Day2_HILIC_3Day2
SA316329MEL_100nM_Timecourse_Day2_HILIC_4Day2
SA316330MEL_100nM_Timecourse_Day2_HILIC_2Day2
SA316331MEL_100nM_Timecourse_Day2_HILIC_1Day2
SA316332MEL_100nM_Timecourse_Day4_HILIC_4Day4
SA316333MEL_100nM_Timecourse_Day4_HILIC_3Day4
SA316334MEL_100nM_Timecourse_Day4_HILIC_1Day4
SA316335MEL_100nM_Timecourse_Day4_HILIC_2Day4
SA316336MEL_100nM_Timecourse_Day6_HILIC_3Day6
SA316337MEL_100nM_Timecourse_Day6_HILIC_1Day6
SA316338MEL_100nM_Timecourse_Day6_HILIC_2Day6
SA316339MEL_100nM_Timecourse_Day8_HILIC_3Day8
SA316340MEL_100nM_Timecourse_Day8_HILIC_2Day8
SA316341MEL_100nM_Timecourse_Day8_HILIC_1Day8
Showing results 1 to 22 of 22

Collection:

Collection ID:CO003018
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:TR003034
Treatment Summary:MEL cells were cultured for up to 8 days in 100 nM folic acid containing RPMI media. The treatments were setup as a reverse time-course experiment and harvested on the same day. Day 0 samples were cultured for 8 days in 2,000 nM folic acid.

Sample Preparation:

Sampleprep ID:SP003031
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 AN004782
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:CH003613
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:MS004528
Analysis ID:AN004782
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
  logo