Summary of Study ST002895
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 | ST002895 |
| Study Title | Polar metabolite levels in K562 cells following folate depletion and inosine supplementation |
| Study Summary | Culture of K562 cells in RPMI media containing 100 nM folic acid for 48hr in the presence or absence of 100 uM inosine followed my metabolomics targeting polar 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 | 2023-10-01 |
| Num Groups | 4 |
| 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: | SU003008 |
| 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 |
|---|---|---|
| SA315542 | K562_100nM_FA_Inosine_HILIC_2 | 100nM_FA_Inosine |
| SA315543 | K562_100nM_FA_Inosine_HILIC_1 | 100nM_FA_Inosine |
| SA315544 | K562_100nM_FA_Inosine_HILIC_3 | 100nM_FA_Inosine |
| SA315545 | K562_100nM_FA_Vehicle_HILIC_3 | 100nM_FA_Vehicle |
| SA315546 | K562_100nM_FA_Vehicle_HILIC_1 | 100nM_FA_Vehicle |
| SA315547 | K562_100nM_FA_Vehicle_HILIC_2 | 100nM_FA_Vehicle |
| SA315548 | K562_2000nM_FA_Inosine_HILIC_3 | 2000nM_FA_Inosine |
| SA315549 | K562_2000nM_FA_Inosine_HILIC_1 | 2000nM_FA_Inosine |
| SA315550 | K562_2000nM_FA_Inosine_HILIC_2 | 2000nM_FA_Inosine |
| SA315551 | K562_2000nM_FA_Vehicle_HILIC_2 | 2000nM_FA_Vehicle |
| SA315552 | K562_2000nM_FA_Vehicle_HILIC_1 | 2000nM_FA_Vehicle |
| SA315553 | K562_2000nM_FA_Vehicle_HILIC_3 | 2000nM_FA_Vehicle |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO003001 |
| 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: | TR003017 |
| Treatment Summary: | K562 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: | SP003014 |
| 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 | AN004754 |
|---|---|
| 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: | CH003586 |
| 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: | MS004500 |
| Analysis ID: | AN004754 |
| 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 |
