Summary of Study ST003022
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 | ST003022 |
| Study Title | Porphyrin metabolite levels in MEL cells following PKC inhibition in 2,000 nM or 100 nM FA conditions |
| Study Summary | Culture of MEL 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 |
| Department | pathology |
| Laboratory | Kanarek Lab |
| Last Name | Kanarek |
| First Name | Naama |
| Address | Enders 1116.2, 300 Longwood Ave, Boston, MA 02115 |
| naama.kanarek@childrens.harvard.edu | |
| Phone | 6173557433 |
| Submit Date | 2023-12-19 |
| Num Groups | 5 |
| 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: | SU003136 |
| 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 | Treatment |
|---|---|---|
| SA327701 | MEL_longterm_PKCi_Porphyrin_100nM_FA_GFX_1 | 100nM_FA_GFX |
| SA327702 | MEL_longterm_PKCi_Porphyrin_100nM_FA_GFX_2 | 100nM_FA_GFX |
| SA327703 | MEL_longterm_PKCi_Porphyrin_100nM_FA_GFX_3 | 100nM_FA_GFX |
| SA327704 | MEL_longterm_PKCi_Porphyrin_100nM_FA_GFX_4 | 100nM_FA_GFX |
| SA327705 | MEL_longterm_PKCi_Porphyrin_100nM_FA_3 | 100nM_FA_Veh |
| SA327706 | MEL_longterm_PKCi_Porphyrin_100nM_FA_4 | 100nM_FA_Veh |
| SA327707 | MEL_longterm_PKCi_Porphyrin_100nM_FA_2 | 100nM_FA_Veh |
| SA327708 | MEL_longterm_PKCi_Porphyrin_100nM_FA_1 | 100nM_FA_Veh |
| SA327709 | MEL_longterm_PKCi_Porphyrin_2000nM_FA_GFX_1 | 2000nM_FA_GFX |
| SA327710 | MEL_longterm_PKCi_Porphyrin_2000nM_FA_GFX_3 | 2000nM_FA_GFX |
| SA327711 | MEL_longterm_PKCi_Porphyrin_2000nM_FA_GFX_2 | 2000nM_FA_GFX |
| SA327712 | MEL_longterm_PKCi_Porphyrin_2000nM_FA_GFX_4 | 2000nM_FA_GFX |
| SA327713 | MEL_longterm_PKCi_Porphyrin_2000nM_FA_2 | 2000nM_FA_Veh |
| SA327714 | MEL_longterm_PKCi_Porphyrin_2000nM_FA_3 | 2000nM_FA_Veh |
| SA327715 | MEL_longterm_PKCi_Porphyrin_2000nM_FA_4 | 2000nM_FA_Veh |
| SA327716 | MEL_longterm_PKCi_Porphyrin_2000nM_FA_1 | 2000nM_FA_Veh |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO003129 |
| 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: | TR003145 |
| Treatment Summary: | Culture of MEL 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 porphyrin metabolites. |
Sample Preparation:
| Sampleprep ID: | SP003142 |
| 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 | AN004956 |
|---|---|
| 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: | CH003740 |
| 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: | MS004696 |
| Analysis ID: | AN004956 |
| 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 |
