Summary of Study ST003075

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 PR001915. The data can be accessed directly via it's Project DOI: 10.21228/M86137 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	ST003075
Study Title	HZV029 TwoPhase Metabolomics and Lipidomics
Study Summary	In this study, a subset of plasma samples were processed using an in-house two phase extraction protocol based on the protocol originally proposed by Matayash et al. The resulting non-polar layer was then analyzed on a Thermo ScientificTM TranscendTM Duo LX-2 UHPLC system interfaced with high resolution Thermo ScientificTM Orbitrap ID-XTM TribidTM mass spectrometer with a HESI ionization source. This dataset was collected as a single batch and was used for the pipeline's ability to detect failed injections, which in this study, was simulated through the substitution of an empty vial for a missing study sample.
Institute	Jackson Laboratory for Genomic Medicine
Laboratory	Shuzhao Li Laboratory
Last Name	Joshua
First Name	Mitchell
Address	10 Discovery Dr, Farmington CT 06032
Email	joshua.mitchell@jax.org
Phone	8608372474
Submit Date	2024-02-15
Publications	Common data models to streamline metabolomics processing and annotation, and implementation in a Python pipeline Joshua Mitchell, Yuanye Chi, Maheshwor Thapa, Zhiqiang Pang, Jianguo Xia, Shuzhao Li doi: https://doi.org/10.1101/2024.02.13.580048
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2024-05-24
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP003196
Sampleprep Summary:	Plasma samples were extracted following an in-house two-phase extraction protocol using methyl tert-butyl ether (MTBE) and methanol as a non-polar and polar extraction solvent and water as a phase separation solvent. The polar phase was used for metabolomics and non-polar phase for lipidomics studies. The MTBE method was introduced by Matayash 2008 which is modified and optimized. Briefly, 100 ml of ice-cold methanol and 300 ml of ice-cold MTBE was added each PBMC pellet to extract polar and non-polar lipid molecules, respectively. For a phase separation, 100 ml of ice-cold water was used. 4 μl of IS solution prepared by mixing 0.12 mL of 1 M D-Glucose-13C6, 0.2 mL of 5 mM Caffeine-3-methyl-13C, 0.6 mL of 10 mM L-Methionine-13C5, 0.6 mL of 20 mM L-Glutamic acid-13C5, 0.8 mL of 10 mM Uracil-15N2, 4 mL of 2 mM L-Tyrosine-15N and 3.68 mL water in 15-mL tube was added in each sample as spike-in controls for polar phase extraction. 10 ml of stable isotope labeled standards (Avanti SPLASH Lipidomix), representing differing lipid classes, were added to each sample to use as a spike ins quality control for non-polar phase extraction. All samples were vortexed (Vortex-Genie 2, Scientific Industries, cat. no. SI-0236) and incubated with shaking ((Eppendorf Thermo Mixer C) at 1000 rpm for 20 min at 4 °C followed by centrifugation at 4 °C for 15 min at 20,817 × g (Centrifuge 5430 R, Eppendorf). 100 ml of lower polar phase for metabolomics were transferred to 1.5 mL autosampler vial and 3 ml injected directly into UHPLC-MS. 250 ml of upper non-polar MTBE phase was transferred to new tube and dried for 2 hrs at Labconco CentriVap Centrifugal Vacuum Concentrator (CentriVap Benchtop Centrifugal Vacuum Concentrator with acrylic lid, Labconco Corporation, cat. no. 7810010), followed by resuspending in 70 ml of methanol:toulene (8:1, v/v) solution. 3 ml of reconstituted solution was injected into UHPLC-MS. For quality control (QC) and assurance (QA), 10 ml of methanol extract from each sample of batch 1 was collected and pooled together to prepare QC sample for polar phase metabolomics. Similarly, 10 ml of reconstituted methanol:toluene (8:1, v/v) solution from each sample of batch 1 was collected and pooled together to prepare QC sample to be used for non-polar phase lipidomics.

Sampleprep ID:

SP003196

Sampleprep Summary:

Plasma samples were extracted following an in-house two-phase extraction protocol using methyl tert-butyl ether (MTBE) and methanol as a non-polar and polar extraction solvent and water as a phase separation solvent. The polar phase was used for metabolomics and non-polar phase for lipidomics studies. The MTBE method was introduced by Matayash 2008 which is modified and optimized. Briefly, 100 ml of ice-cold methanol and 300 ml of ice-cold MTBE was added each PBMC pellet to extract polar and non-polar lipid molecules, respectively. For a phase separation, 100 ml of ice-cold water was used. 4 μl of IS solution prepared by mixing 0.12 mL of 1 M D-Glucose-13C6, 0.2 mL of 5 mM Caffeine-3-methyl-13C, 0.6 mL of 10 mM L-Methionine-13C5, 0.6 mL of 20 mM L-Glutamic acid-13C5, 0.8 mL of 10 mM Uracil-15N2, 4 mL of 2 mM L-Tyrosine-15N and 3.68 mL water in 15-mL tube was added in each sample as spike-in controls for polar phase extraction. 10 ml of stable isotope labeled standards (Avanti SPLASH Lipidomix), representing differing lipid classes, were added to each sample to use as a spike ins quality control for non-polar phase extraction. All samples were vortexed (Vortex-Genie 2, Scientific Industries, cat. no. SI-0236) and incubated with shaking ((Eppendorf Thermo Mixer C) at 1000 rpm for 20 min at 4 °C followed by centrifugation at 4 °C for 15 min at 20,817 × g (Centrifuge 5430 R, Eppendorf). 100 ml of lower polar phase for metabolomics were transferred to 1.5 mL autosampler vial and 3 ml injected directly into UHPLC-MS. 250 ml of upper non-polar MTBE phase was transferred to new tube and dried for 2 hrs at Labconco CentriVap Centrifugal Vacuum Concentrator (CentriVap Benchtop Centrifugal Vacuum Concentrator with acrylic lid, Labconco Corporation, cat. no. 7810010), followed by resuspending in 70 ml of methanol:toulene (8:1, v/v) solution. 3 ml of reconstituted solution was injected into UHPLC-MS. For quality control (QC) and assurance (QA), 10 ml of methanol extract from each sample of batch 1 was collected and pooled together to prepare QC sample for polar phase metabolomics. Similarly, 10 ml of reconstituted methanol:toluene (8:1, v/v) solution from each sample of batch 1 was collected and pooled together to prepare QC sample to be used for non-polar phase lipidomics.