Summary of Study ST001613

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 PR001033. The data can be accessed directly via it's Project DOI: 10.21228/M85M45 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001613
Study Title	Comparing gas chromatography with time-of-flight, quadrupole time-of-light and quadrupole mass spectrometry for stable isotope tracing (part-II)
Study Summary	Stable isotope tracers are applied in vivo and in vitro studies to reveal the activity of enzymes and intracellular metabolic pathways. Most often, such tracers are used with gas chromatography coupled to mass spectrometry (GC-MS) due to its ease of operation and reproducible mass spectral databases. Differences in isotope tracer performance of classic GC-quadrupole MS instrument and newer time-of-flight instruments are not well-studied. Here, we used three commercially available instruments for the analysis of identical samples from a stable isotope labeling study that used [U-13C6] d-glucose to investigate the metabolism of Rothia mucilaginosa with respect to 29 amino acids and hydroxyl acids involved in primary metabolism. Overall, all three GC-MS instruments (low-resolution GC-SQ-MS, low-resolution GC-TOF-MS, and high-resolution GC-Q-TOF-MS) can be used to perform stable isotope tracing studies for glycolytic intermediates, TCA metabolites and amino acids, yielding similar biological results, with high-resolution GC-Q-TOF-MS offering additional capabilities to identify chemical structures of unknown compounds that might show significant isotope enrichments in biological studies.
Institute	University of California, Davis
Last Name	Zhang
First Name	Ying
Address	451 East Health Science Drive, Davis, CA, 95616, USA
Email	ythzhang@ucdavis.edu
Phone	1-530-752-8129
Submit Date	2020-11-05
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	GC-MS
Release Date	2020-12-10
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP001696
Sampleprep Summary:	For the 29 unlabeled standards mixture, 10 µL of the final solution was dried down for GC-MS measurement. Derivatization and data acquisition of mixture aliquots by GC-MS were reproduced on three days for inter-day precision. Dried mixtures were derivatized by adding 10 µL of 40 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and shaking at 30°C for 1.5 h. Subsequently, 90 µL MTBSTFA (Sigma-Aldrich, St. Louis, MO, USA) was added with 13 fatty acid methyl esters (FAMEs) as retention index markers and shaken at 80°C for 30 min. Samples were immediately transferred to crimp top vials and injected onto each GC-MS instrument. Same samples of R. mucilaginosa cultures were extracted using published methods [19]. Samples were added 1 mL pre-chilled, degassed acetonitrile: isopropanol: water (v/v/v 3:3:2, Fisher Scientific) followed by vortexing 30 s and shaking at 4°C for 5 min. Samples were centrifuged for 2 min at 12,210 × g to precipitate debris from extracts. Supernatants were collected and split into two equal portions. One aliquot was dried to completeness in a Labconco cold trap centrifuge evaporator and then resuspended in 0.5 mL degassed acetonitrile: water (v/v 1:1, Fisher Scientific) to remove triacylglycerides. Resuspension solutions were vortexed for 30s and centrifuged for 2 min. Supernatants were transferred into clean Eppendorf tubes and dried down completely. Dried extracts were derivatized as given above.

Sampleprep ID:

SP001696

Sampleprep Summary:

For the 29 unlabeled standards mixture, 10 µL of the final solution was dried down for GC-MS measurement. Derivatization and data acquisition of mixture aliquots by GC-MS were reproduced on three days for inter-day precision. Dried mixtures were derivatized by adding 10 µL of 40 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and shaking at 30°C for 1.5 h. Subsequently, 90 µL MTBSTFA (Sigma-Aldrich, St. Louis, MO, USA) was added with 13 fatty acid methyl esters (FAMEs) as retention index markers and shaken at 80°C for 30 min. Samples were immediately transferred to crimp top vials and injected onto each GC-MS instrument. Same samples of R. mucilaginosa cultures were extracted using published methods [19]. Samples were added 1 mL pre-chilled, degassed acetonitrile: isopropanol: water (v/v/v 3:3:2, Fisher Scientific) followed by vortexing 30 s and shaking at 4°C for 5 min. Samples were centrifuged for 2 min at 12,210 × g to precipitate debris from extracts. Supernatants were collected and split into two equal portions. One aliquot was dried to completeness in a Labconco cold trap centrifuge evaporator and then resuspended in 0.5 mL degassed acetonitrile: water (v/v 1:1, Fisher Scientific) to remove triacylglycerides. Resuspension solutions were vortexed for 30s and centrifuged for 2 min. Supernatants were transferred into clean Eppendorf tubes and dried down completely. Dried extracts were derivatized as given above.