Summary of Study ST003021

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 IDST003021
Study TitlePorphyrin metabolite levels in K562 cells following PKC inhibition in 2,000 nM or 100 nM FA conditions
Study SummaryCulture of K562 cells in RPMI media containing 2000 nM or 100 nM folic acid for 6 days in the presence or absence of the PKC inhibitor, GF109203X. These samples were followed up by metabolomics targeting porphyrin 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 Date2023-12-19
Num Groups5
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:SU003135
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
SA327685K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_1100nM_FA_GFX
SA327686K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_2100nM_FA_GFX
SA327687K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_3100nM_FA_GFX
SA327688K562_longterm_PKCi_Porphyrin_100nM_FA_GFX_4100nM_FA_GFX
SA327689K562_longterm_PKCi_Porphyrin_100nM_FA_3100nM_FA_Veh
SA327690K562_longterm_PKCi_Porphyrin_100nM_FA_4100nM_FA_Veh
SA327691K562_longterm_PKCi_Porphyrin_100nM_FA_2100nM_FA_Veh
SA327692K562_longterm_PKCi_Porphyrin_100nM_FA_1100nM_FA_Veh
SA327693K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_12000nM_FA_GFX
SA327694K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_32000nM_FA_GFX
SA327695K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_22000nM_FA_GFX
SA327696K562_longterm_PKCi_Porphyrin_2000nM_FA_GFX_42000nM_FA_GFX
SA327697K562_longterm_PKCi_Porphyrin_2000nM_FA_22000nM_FA_Veh
SA327698K562_longterm_PKCi_Porphyrin_2000nM_FA_32000nM_FA_Veh
SA327699K562_longterm_PKCi_Porphyrin_2000nM_FA_42000nM_FA_Veh
SA327700K562_longterm_PKCi_Porphyrin_2000nM_FA_12000nM_FA_Veh
Showing results 1 to 16 of 16

Collection:

Collection ID:CO003128
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:TR003144
Treatment Summary:Culture of K562 cells for 6 days in RPMI media containing 2000 nM or 100 nM folic acid for 6 days in the presence or absence of the PKC inhibitor, GF109203X. These samples were followed up by metabolomics targeting polar metabolites.

Sample Preparation:

Sampleprep ID:SP003141
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 AN004955
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 Arbitrary Units

Chromatography:

Chromatography ID:CH003739
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: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.
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:MS004695
Analysis ID:AN004955
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
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