Summary of Study ST001812

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 PR001145. The data can be accessed directly via it's Project DOI: 10.21228/M8Q12H 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.

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Study ID	ST001812
Study Title	Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a glutaminase in roots of Arabidopsis thaliana (part II)
Study Type	Targeted Metabolite Quantification
Study Summary	In this study, we used Arabidopsis root extracts, spiked with amide nitrogen labeled (15N1) Glutamine and a purified recombinant protein, both full length and glutaminase domain only versions, to determine the amido group acceptor, if any, in the glutamine amidotransferase reaction.
Institute	Agriculture and Agri-Food Canada
Department	London Research and Development Centre
Laboratory	Frederic Marsolais
Last Name	Kambhampati
First Name	Shrikaar
Address	1391 Sandford St, London, ON N5V 4T3, Canada
Email	shrikaar.k@gmail.com
Phone	3144025550
Submit Date	2021-06-01
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2021-06-16
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP001895
Sampleprep Summary:	Fifty mg of root tissue was excised from 10 day old seedlings of Arabidopsis grown with 5 mM KNO3, collected in 2 mL Eppendorf tubes and flash frozen in liquid N2. Frozen tissue was homogenized using a tissue lyser and metabolites were isolated using 1 mL of methanol:water (4:1) with incubation in an ultra-sonication bath for 20 min followed by shaking for 30 min at 4 °C. The mixture was centrifuged at 12,000 × g for 10 min at 4 °C and 700 µl of the supernatant was transferred into fresh tubes and evaporated to dryness using a Vacufuge at ambient temperature. Dried metabolite extracts were re-suspended in HEPES buffer pH 7.5 instead of 1:1 methanol:water. Samples were spiked with a final concentration of 1 µM 15N Gln and 5 µg of the full length or glutaminase domain versions of recombinant GAT1_2.1 protein along with 2 mM DTT and 5 mM MgCl2. Following this, samples were incubated at 37 °C for 2 hours and then filtered through a 3K micro centrifuge filter (Sigma-Aldrich) to remove the protein. Samples were then evaporated to dryness using a vacufuge at ambient temperature and the residue was re-dissolved in 1:1 methanol:water, filtered with a 0.2 µm PTFE microfuge filters (Whatman) and subjected to LC-MS analysis and ammonium quantification.

Sampleprep ID:

SP001895

Sampleprep Summary:

Fifty mg of root tissue was excised from 10 day old seedlings of Arabidopsis grown with 5 mM KNO3, collected in 2 mL Eppendorf tubes and flash frozen in liquid N2. Frozen tissue was homogenized using a tissue lyser and metabolites were isolated using 1 mL of methanol:water (4:1) with incubation in an ultra-sonication bath for 20 min followed by shaking for 30 min at 4 °C. The mixture was centrifuged at 12,000 × g for 10 min at 4 °C and 700 µl of the supernatant was transferred into fresh tubes and evaporated to dryness using a Vacufuge at ambient temperature. Dried metabolite extracts were re-suspended in HEPES buffer pH 7.5 instead of 1:1 methanol:water. Samples were spiked with a final concentration of 1 µM 15N Gln and 5 µg of the full length or glutaminase domain versions of recombinant GAT1_2.1 protein along with 2 mM DTT and 5 mM MgCl2. Following this, samples were incubated at 37 °C for 2 hours and then filtered through a 3K micro centrifuge filter (Sigma-Aldrich) to remove the protein. Samples were then evaporated to dryness using a vacufuge at ambient temperature and the residue was re-dissolved in 1:1 methanol:water, filtered with a 0.2 µm PTFE microfuge filters (Whatman) and subjected to LC-MS analysis and ammonium quantification.