Summary of Study ST002992

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 IDST002992
Study TitlePolar metabolite levels in K562 cells following C13-Serine and C13-Glycine tracing
Study SummaryCulture 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 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 Date2023-10-02
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

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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:SU003105
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
SA325810K562_100nM_FA_C13_Glycine_HILIC_4100nM_FA_C13_Glycine
SA325811K562_100nM_FA_C13_Glycine_HILIC_1100nM_FA_C13_Glycine
SA325812K562_100nM_FA_C13_Glycine_HILIC_2100nM_FA_C13_Glycine
SA325813K562_100nM_FA_C13_Glycine_HILIC_3100nM_FA_C13_Glycine
SA325814K562_100nM_FA_C13_Serine_HILIC_1100nM_FA_C13_Serine
SA325815K562_100nM_FA_C13_Serine_HILIC_2100nM_FA_C13_Serine
SA325816K562_100nM_FA_C13_Serine_HILIC_4100nM_FA_C13_Serine
SA325817K562_100nM_FA_C13_Serine_HILIC_3100nM_FA_C13_Serine
SA325818K562_100nM_FA_Unlabeled_HILIC_3100nM_FA_Unlabeled
SA325819K562_100nM_FA_Unlabeled_HILIC_1100nM_FA_Unlabeled
SA325820K562_100nM_FA_Unlabeled_HILIC_2100nM_FA_Unlabeled
SA325821K562_100nM_FA_Unlabeled_HILIC_4100nM_FA_Unlabeled
SA325822K562_2000nM_FA_C13_Glycine_HILIC_12000nM_FA_C13_Glycine
SA325823K562_2000nM_FA_C13_Glycine_HILIC_22000nM_FA_C13_Glycine
SA325824K562_2000nM_FA_C13_Glycine_HILIC_32000nM_FA_C13_Glycine
SA325825K562_2000nM_FA_C13_Glycine_HILIC_42000nM_FA_C13_Glycine
SA325826K562_2000nM_FA_C13_Serine_HILIC_12000nM_FA_C13_Serine
SA325827K562_2000nM_FA_C13_Serine_HILIC_42000nM_FA_C13_Serine
SA325828K562_2000nM_FA_C13_Serine_HILIC_22000nM_FA_C13_Serine
SA325829K562_2000nM_FA_C13_Serine_HILIC_32000nM_FA_C13_Serine
SA325830K562_2000nM_FA_Unlabeled_HILIC_22000nM_FA_Unlabeled
SA325831K562_2000nM_FA_Unlabeled_HILIC_32000nM_FA_Unlabeled
SA325832K562_2000nM_FA_Unlabeled_HILIC_12000nM_FA_Unlabeled
SA325833K562_2000nM_FA_Unlabeled_HILIC_42000nM_FA_Unlabeled
Showing results 1 to 24 of 24

Collection:

Collection ID:CO003098
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:TR003114
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:SP003111
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 AN004912
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:CH003707
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:MS004655
Analysis ID:AN004912
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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