Summary of Study ST002894

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	ST002894
Study Title	Folate levels in MEL cells following folate depletion
Study Summary	Culture of MEL cells in RPMI media containing 100 nM folic acid for up to 8 days followed my metabolomics targeting folate metabolites. All samples were harvested at the same time and the study was set up as a reverse time-course. Day 0 samples were cultured in 2,000 nM folate for the full 8 days.
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	2023-10-01
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

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Sample Preparation:

Sampleprep ID:	SP003013
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:

SP003013

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.