Summary of Study ST000609

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 PR000446. The data can be accessed directly via it's Project DOI: 10.21228/M8DC72 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	ST000609
Study Title	CHEAR Plasma Reference Material
Study Type	Broad spectrum, reverse phase LCMS metabolomics
Study Summary	CHEAR PlasmaRef_20160726 was provided by Emory University. The material was prepared and analyzed using the Metabolomics Reverse Phase Broad Spectrum analysis on a Synapt G2-Si system. Six samples were injected of the sample reference material that were prepared in replicate.
Institute	RTI International
Department	ACP
Last Name	Fennell
First Name	Timothy
Address	3040 E Cornwallis Rd, Durham, North Carolina, 27709, USA
Email	fennell@rti.org
Phone	9194852781
Submit Date	2017-05-16
Num Groups	1
Total Subjects	6
Raw Data Available	Yes
Raw Data File Type(s)	raw(Waters)
Chear Study	Yes
Analysis Type Detail	LC-MS
Release Date	2024-12-31
Release Version	1

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Project:

Project ID:	PR000446
Project DOI:	doi: 10.21228/M8DC72
Project Title:	CHEAR Plasma Reference Material
Project Type:	Broad spectrum, reverse phase LCMS metabolomics
Project Summary:	CHEAR PlasmaRef_20160726 was provided by Emory University. The material was prepared and analyzed using the Metabolomics Reverse Phase Broad Spectrum analysis on a Synapt G2-Si system. Six samples were injected of the sample reference material that were prepared in replicate.
Institute:	RTI International
Department:	APC
Last Name:	Fennell
First Name:	Timothy
Address:	3040 E Cornwallis Rd, Durham, North Carolina, 27709, USA
Email:	fennell@rti.org
Phone:	9194852781
Funding Source:	U2CES026544

Subject:

Subject ID:	SU000632
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Subject Comments:	Plasma
Species Group:	Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Matrix
SA033586	PP_A_09_12	Plasma
SA033587	PP_A_09_11	Plasma
SA033588	PP_A_09_10	Plasma
SA033589	PP_A_09_8	Plasma
SA033590	PP_A_09_9	Plasma
SA033591	PP_A_09_7	Plasma

Showing results 1 to 6 of 6

Showing results 1 to 6 of 6

Collection:

Collection ID:	CO000626
Collection Summary:	N/A
Sample Type:	Plasma
Storage Conditions:	-80˚C

Treatment:

Treatment ID:	TR000646
Treatment Summary:	N/A

Sample Preparation:

Sampleprep ID:	SP000639
Sampleprep Summary:	Plasma: Parent and Sub-Aliquots The CHEAR PlasmaRef_20160726 (500 mL) was removed from -80°C storage and thawed by storing it at 4°C overnight. The following day, the thawed plasma was inspected to ensure there was no ice remaining and the bulk plasma was then mixed in the 4°C room by repeatedly inverting the container. Parent Aliquots were quickly prepared in the 4°C room using the Drummond pipet aid and serological pipet. 40 mL plasma aliquots were transferred to 50 mL tubes in 4°C room and capped immediately and placed on ice. If needed, the bulk plasma was mixed in between aliquots. The parent aliquots were labeled appropriately. Sub-Aliquots were prepared on ice at the bench. The parent aliquots were mixed by inverting the tube thoroughly before and in between aliquots as needed. 1 mL plasma aliquots were transferred to cryovials and capped immediately and stored on ice until sample splitting was competed. The sub aliquots were labeled appropriately. Sub-aliquots were stored at -80°C. Plasma Aliquots for LCMS platforms: The sub-aliquot of PP_A_09 was used to prepare aliquots for various LCMS platforms. PP_A_09 was thawed on ice for 30 – 60 min and vortexed briefly on a vortexer, followed by centrifugation at 4 °C for 2 minutes at 16,000 rcf. Six aliquots of 60 µL volumes were prepared for Reverse Phase analysis from PP_A_09. Aliquots were stored at -80 °C until analysis. Sample preparation for Reverse Phase: The 6 plasma aliquots were thawed on ice for 30–60 min, and then 50 µL of each sample was transferred to a new pre-labeled Lo-Bind tube. A volume of 400 µL methanol containing 0.025 mg/mL tryptophan-d5 as the internal standard was added to extract metabolites by vortex for 2 mins at 5000 rpm. Sample solution was centrifuged at 16,000 rcf for 4 min at room temperature to precipitate proteins. A volume of 350 µL of supernatant was transferred to pre-labeled 2.0 mL Lo-bind tubes and dried down using a SpeedVac system (LabConco CentriVap) at room temperature. A volume of 100 µL of Water/Methanol solution (95:5) was added to reconstitute each sample. Samples were then thoroughly mixed by vortex for 10 mins at 5000 rpm and then centrifuged at 16,000 rcf for 4 min at room temperature. The supernatants were transferred to pre-labeled autosampler vials before analysis. LC-MS analysis: The prepared plasma samples were analyzed by an LC-MS system comprised of a Waters Acquity UPLC and a Synapt G2-Si ESI-Q-TOF mass spectrometer. Chromatographic separation was accomplished on an Acquity HSST3 C18 column (2.1 X 100 mm, 1.8 µm) at 50 °C using a gradient elution comprised of mobile phase A: 0.1% formic acid-water (v/v), and mobile phase B: 0.1% formic acid-methanol (v/v), using a flow rate of 0.400 mL/min. The injection volume was 5 µL.

Sampleprep ID:

SP000639

Sampleprep Summary:

Plasma: Parent and Sub-Aliquots The CHEAR PlasmaRef_20160726 (500 mL) was removed from -80°C storage and thawed by storing it at 4°C overnight. The following day, the thawed plasma was inspected to ensure there was no ice remaining and the bulk plasma was then mixed in the 4°C room by repeatedly inverting the container. Parent Aliquots were quickly prepared in the 4°C room using the Drummond pipet aid and serological pipet. 40 mL plasma aliquots were transferred to 50 mL tubes in 4°C room and capped immediately and placed on ice. If needed, the bulk plasma was mixed in between aliquots. The parent aliquots were labeled appropriately. Sub-Aliquots were prepared on ice at the bench. The parent aliquots were mixed by inverting the tube thoroughly before and in between aliquots as needed. 1 mL plasma aliquots were transferred to cryovials and capped immediately and stored on ice until sample splitting was competed. The sub aliquots were labeled appropriately. Sub-aliquots were stored at -80°C. Plasma Aliquots for LCMS platforms: The sub-aliquot of PP_A_09 was used to prepare aliquots for various LCMS platforms. PP_A_09 was thawed on ice for 30 – 60 min and vortexed briefly on a vortexer, followed by centrifugation at 4 °C for 2 minutes at 16,000 rcf. Six aliquots of 60 µL volumes were prepared for Reverse Phase analysis from PP_A_09. Aliquots were stored at -80 °C until analysis. Sample preparation for Reverse Phase: The 6 plasma aliquots were thawed on ice for 30–60 min, and then 50 µL of each sample was transferred to a new pre-labeled Lo-Bind tube. A volume of 400 µL methanol containing 0.025 mg/mL tryptophan-d5 as the internal standard was added to extract metabolites by vortex for 2 mins at 5000 rpm. Sample solution was centrifuged at 16,000 rcf for 4 min at room temperature to precipitate proteins. A volume of 350 µL of supernatant was transferred to pre-labeled 2.0 mL Lo-bind tubes and dried down using a SpeedVac system (LabConco CentriVap) at room temperature. A volume of 100 µL of Water/Methanol solution (95:5) was added to reconstitute each sample. Samples were then thoroughly mixed by vortex for 10 mins at 5000 rpm and then centrifuged at 16,000 rcf for 4 min at room temperature. The supernatants were transferred to pre-labeled autosampler vials before analysis. LC-MS analysis: The prepared plasma samples were analyzed by an LC-MS system comprised of a Waters Acquity UPLC and a Synapt G2-Si ESI-Q-TOF mass spectrometer. Chromatographic separation was accomplished on an Acquity HSST3 C18 column (2.1 X 100 mm, 1.8 µm) at 50 °C using a gradient elution comprised of mobile phase A: 0.1% formic acid-water (v/v), and mobile phase B: 0.1% formic acid-methanol (v/v), using a flow rate of 0.400 mL/min. The injection volume was 5 µL.

Chromatography:

Chromatography ID:	CH000665
Chromatography Summary:	Reversed-Phase Gradient Seperation
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity HSS T3 (100 x 2.1mm,1.8um)
Column Pressure:	6,000-10,000 psi
Column Temperature:	50 °C
Flow Rate:	0.4 mL/min
Injection Temperature:	8 °C
Internal Standard:	L-Tryptophan-d5
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% methanol; 0.1% formic acid
Analytical Time:	22 min
Weak Wash Solvent Name:	10% MeOH
Weak Wash Volume:	1000 µL
Strong Wash Solvent Name:	80% MeOH
Strong Wash Volume:	1000 µL
Target Sample Temperature:	8 °C
Sample Loop Size:	10 µL
Sample Syringe Size:	100 µL
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN000932
Analysis Type:	MS
Instrument Name:	Waters Synapt-G2-Si
Chromatography ID:	CH000665
Num Factors:	1
Rt Units:	Minutes
Results File:	ST000609_AN000932_Results.txt
Units:	Normalized Abundance

Analysis ID:	AN000933
Analysis Type:	MS
Instrument Name:	Waters Synapt-G2-Si
Chromatography ID:	CH000665
Num Factors:	1
Rt Units:	Minutes
Results File:	ST000609_AN000933_Results.txt
Units:	Normalized Abundance