Summary of Study ST003198

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 PR001988. The data can be accessed directly via it's Project DOI: 10.21228/M8RJ07 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	ST003198
Study Title	UQ/RQ panel on human tissue
Study Summary	The extraction of nonpolar metabolites from the tissues of human for analysis by LCMS to measure levels of Ubiquinone and Rhodoquinone.
Institute	UMass Chan Medical School
Department	Program in Molecular Medicine
Laboratory	Spinelli Lab
Last Name	Jerome
First Name	Madison
Address	55 N Lake Ave, Worcester, MA 01655
Email	madison.jerome@umassmed.edu
Phone	(508) 856-8989 ext. 68148
Submit Date	2024-05-01
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-02-04
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP003324
Sampleprep Summary:	UQRQ isolation from tissues: Tissues were flash frozen in liquid nitrogen and powderized using a mortar and pestle. Approximately 10 mg of tissue powder was transferred into an Eppendorf tube. The tissue powder was re-suspended in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl and then vortexed for 15 minutes at 4°C. 500 µL LCMS-grade hexane (Honeywell) was added to the lysate and again vortexed for 15 minutes at 4°C. Samples were then centrifuged at 16,000 x g for 10 minutes at 4°C with the top (hexane) layer containing UQ and RQ, the middle layer containing the acidified methanol, and the bottom layer containing the protein. The top layer was transferred into a new Eppendorf tube, dried down in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco), and stored at -80°C until they were re-suspended for LC-MS analysis. The acidified methanol layer was discarded, and the bottom protein layer was saved for protein quantification. The protein layer from the metabolite isolation was re-suspended in 1 mL of RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 0.1% SDS, 1% Triton-X 100 (Sigma), 0.5% deoxycholate (Sigma), cOmplete EDTA-free protease inhibitor (Sigma)). The samples were vortexed for 10 minutes at 4°C and then centrifuged at 16,000 x g for 10 minutes at 4°C. Protein concentrations for each sample were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/µL ) concentration. Isolation of mitochondria from tissues: Purified mitochondria pellets were subsequently vortexed in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl for 10 minutes at 4°C. 500 µL of 100% LCMS-grade hexane (Honeywell) was added to each tube and vortexed for an additional 10 minutes at 4°C. Samples were then centrifuged for 10 minutes at 21,300 x g at 4°C. The top (hexane) layer of the sample (containing UQ and RQ) was transferred to a new labeled 1.5 mL Eppendorf tube and dried in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). Samples were stored in -80°C freezer prior to resuspension and running on LC-MS. The bottom layer was discarded and the pellet was saved for protein quantification. Protein was isolated as described above. Protein concentrations were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/uL) concentration. Isolation of UQ/RQ from purified mitochondria: Purified mitochondria pellets were subsequently vortexed in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl for 10 minutes at 4°C. 500 µL of 100% LCMS-grade hexane (Honeywell) was added to each tube and vortexed for an additional 10 minutes at 4°C. Samples were then centrifuged for 10 minutes at 21,300 x g at 4°C. The top (hexane) layer of the sample (containing UQ and RQ) was transferred to a new labeled 1.5 mL Eppendorf tube and dried in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). Samples were stored in -80°C freezer prior to resuspension and running on LC-MS. The bottom layer was discarded and the pellet was saved for protein quantification. Protein was isolated as described above. Protein concentrations were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/uL) concentration.

Sampleprep ID:

SP003324

Sampleprep Summary:

UQRQ isolation from tissues: Tissues were flash frozen in liquid nitrogen and powderized using a mortar and pestle. Approximately 10 mg of tissue powder was transferred into an Eppendorf tube. The tissue powder was re-suspended in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl and then vortexed for 15 minutes at 4°C. 500 µL LCMS-grade hexane (Honeywell) was added to the lysate and again vortexed for 15 minutes at 4°C. Samples were then centrifuged at 16,000 x g for 10 minutes at 4°C with the top (hexane) layer containing UQ and RQ, the middle layer containing the acidified methanol, and the bottom layer containing the protein. The top layer was transferred into a new Eppendorf tube, dried down in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco), and stored at -80°C until they were re-suspended for LC-MS analysis. The acidified methanol layer was discarded, and the bottom protein layer was saved for protein quantification. The protein layer from the metabolite isolation was re-suspended in 1 mL of RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 0.1% SDS, 1% Triton-X 100 (Sigma), 0.5% deoxycholate (Sigma), cOmplete EDTA-free protease inhibitor (Sigma)). The samples were vortexed for 10 minutes at 4°C and then centrifuged at 16,000 x g for 10 minutes at 4°C. Protein concentrations for each sample were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/µL ) concentration. Isolation of mitochondria from tissues: Purified mitochondria pellets were subsequently vortexed in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl for 10 minutes at 4°C. 500 µL of 100% LCMS-grade hexane (Honeywell) was added to each tube and vortexed for an additional 10 minutes at 4°C. Samples were then centrifuged for 10 minutes at 21,300 x g at 4°C. The top (hexane) layer of the sample (containing UQ and RQ) was transferred to a new labeled 1.5 mL Eppendorf tube and dried in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). Samples were stored in -80°C freezer prior to resuspension and running on LC-MS. The bottom layer was discarded and the pellet was saved for protein quantification. Protein was isolated as described above. Protein concentrations were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/uL) concentration. Isolation of UQ/RQ from purified mitochondria: Purified mitochondria pellets were subsequently vortexed in 500 µL of 99.9% LCMS-grade methanol (Fisher) 0.1% HCl for 10 minutes at 4°C. 500 µL of 100% LCMS-grade hexane (Honeywell) was added to each tube and vortexed for an additional 10 minutes at 4°C. Samples were then centrifuged for 10 minutes at 21,300 x g at 4°C. The top (hexane) layer of the sample (containing UQ and RQ) was transferred to a new labeled 1.5 mL Eppendorf tube and dried in a Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). Samples were stored in -80°C freezer prior to resuspension and running on LC-MS. The bottom layer was discarded and the pellet was saved for protein quantification. Protein was isolated as described above. Protein concentrations were calculated using the Pierce BCA Protein Assay Kit (Life Technologies). The protein concentrations were used to calculate the re-suspension volume to normalize all samples for LC-MS analysis to equal (20 µg/uL) concentration.