Summary of Study ST002907

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	ST002907
Study Title	Folate levels in K562 cells following short-term folate depletion
Study Summary	Culture of K562 cells for 48hr in RPMI media containing 2000 nM, 100 nM, 50 nM, 20 nM, or 0 nM folic acid followed by folate metabolomics.
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	6173557433
Submit Date	2023-10-02
Num Groups	6
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:	SU003020
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
SA315960	K562_Titration_0nM_folate_1	0_nM_FA
SA315961	K562_Titration_0nM_folate_3	0_nM_FA
SA315962	K562_Titration_0nM_folate_2	0_nM_FA
SA315963	K562_Titration_100nM_folate_2	100_nM_FA
SA315964	K562_Titration_100nM_folate_1	100_nM_FA
SA315965	K562_Titration_100nM_folate_3	100_nM_FA
SA315966	K562_Titration_2000nM_folate_2	2000_nM_FA
SA315967	K562_Titration_2000nM_folate_1	2000_nM_FA
SA315968	K562_Titration_2000nM_folate_3	2000_nM_FA
SA315969	K562_Titration_20nM_folate_3	20_nM_FA
SA315970	K562_Titration_20nM_folate_1	20_nM_FA
SA315971	K562_Titration_20nM_folate_2	20_nM_FA
SA315972	K562_Titration_50nM_folate_2	50_nM_FA
SA315973	K562_Titration_50nM_folate_1	50_nM_FA
SA315974	K562_Titration_50nM_folate_3	50_nM_FA

Showing results 1 to 15 of 15

Showing results 1 to 15 of 15

Collection:

Collection ID:	CO003013
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:	TR003029
Treatment Summary:	Culture of K562 cells for 48hr in RPMI media containing 2000 nM, 100 nM, 50 nM, 20 nM, or 0 nM folic acid followed by folate metabolomics.

Sample Preparation:

Sampleprep ID:	SP003026
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 folate metabolite detection. Folate samples were resuspended in reconstitution buffer (0.5% ascorbic acid, 1% K2HPO4 and 0.5% 2-mercaptoethanol) containing rat serum (Sigma, R9759). The rat serum contains the enzymes required to strip the polyglutamate tail from the measured folate. Rat endogenous folate was removed from the rat serum by activated carbon treatment (Sigma, C9157). Polyglutamate tail removal was carried out for 2 h at 37 °C. After the polyglutamate tail removal, the pH was adjusted to 4 using formic acid and samples were loaded on Bond Elute-pH columns (Agilent, 14102062) for a pH-based clean-up. Columns were eluted with 300 μl elution buffer (50% methanol, 0.25% NH4OAc, 0.5% 2-mercaptoethanol), followed by drying the samples using a liquid nitrogen dryer manifold and resuspension in 25 μl water.

Sampleprep ID:

SP003026

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 folate metabolite detection. Folate samples were resuspended in reconstitution buffer (0.5% ascorbic acid, 1% K2HPO4 and 0.5% 2-mercaptoethanol) containing rat serum (Sigma, R9759). The rat serum contains the enzymes required to strip the polyglutamate tail from the measured folate. Rat endogenous folate was removed from the rat serum by activated carbon treatment (Sigma, C9157). Polyglutamate tail removal was carried out for 2 h at 37 °C. After the polyglutamate tail removal, the pH was adjusted to 4 using formic acid and samples were loaded on Bond Elute-pH columns (Agilent, 14102062) for a pH-based clean-up. Columns were eluted with 300 μl elution buffer (50% methanol, 0.25% NH4OAc, 0.5% 2-mercaptoethanol), followed by drying the samples using a liquid nitrogen dryer manifold and resuspension in 25 μl water.

Combined analysis:

Analysis ID	AN004770
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	UNSPECIFIED
Units	peak area

Chromatography:

Chromatography ID:	CH003602
Chromatography Summary:	5 μl 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). The column oven and autosampler tray were held at 30 °C and 4 °C, respectively. The following conditions were used to achieve chromatographic separation: buffer A was 0.1% formic acid; buffer B was acetonitrile with 0.1% formic acid. The chromatographic gradient was run at a flow rate of 0.250 ml min−1 as follows: 0–5 min: gradient was held at 5% B; 5–10 min: linear gradient of 5% to 36% B; 10.1–14.0 min: linear gradient from 36–95% B; 14.1–18.0 min: gradient was returned to 5% B.
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 3mm,2.6um)
Column Temperature:	30
Flow Gradient:	0.250 ml min−1 as follows: 0–5 min: gradient was held at 5% B; 5–10 min: linear gradient of 5% to 36% B; 10.1–14.0 min: linear gradient from 36–95% B; 14.1–18.0 min: gradient was returned to 5% B.
Flow Rate:	250 uL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004516
Analysis ID:	AN004770
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 three narrow-range scans: 438–450 m/z; 452–462 m/z; and 470–478 m/z, with the resolution set at 70,000, the AGC target at 10e6, and the maximum injection time of 150 ms. HESI settings were: sheath gas flow rate: 40; Aux gas flow rate: 10; Sweep gas: 0; Spray voltage: 2.8 (neg) 3.5 (pos); Capillary temperature 300; S-lens RF level 50; Aux gas heater temp: 350.
Ion Mode:	UNSPECIFIED