Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA303601

Local Sample ID:	2907927
Subject ID:	SU002928
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	18 and older
Gender:	Male and female

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Subject:

Subject ID:	SU002928
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	18 and older
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
2907927	SA303601	FL036650	MDRD	study

Collection:

Collection ID:	CO002921
Collection Summary:	samples were obtained from NIDDK repository
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR002937
Treatment Summary:	Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.

Treatment ID:

TR002937

Treatment Summary:

Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.

Sample Preparation:

Sampleprep ID:	SP002934
Sampleprep Summary:	All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions, however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z. Raw data files are archived and extracted as described below

Sampleprep ID:

SP002934

Sampleprep Summary:

All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions, however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z. Raw data files are archived and extracted as described below

Combined analysis:

Analysis ID	AN004590	AN004591	AN004592	AN004593
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	Reversed phase	HILIC
Chromatography system	Waters Acquity	Waters Acquity	Waters Acquity	Waters Acquity
Column	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	POSITIVE	NEGATIVE	NEGATIVE
Units	raw area counts rescaled to set the median equal to 1	raw area counts rescaled to set the median equal to 1	raw area counts rescaled to set the median equal to 1	raw area counts rescaled to set the median equal to 1

Chromatography:

Chromatography ID:	CH003450
Chromatography Summary:	Low pH polar (LC/MS Pos early)
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	-
Flow Gradient:	-
Flow Rate:	-
Solvent A:	-
Solvent B:	-
Chromatography Type:	Reversed phase

Chromatography ID:	CH003451
Chromatography Summary:	Low pH Lipophilic (LC/MS Pos late)
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	-
Flow Gradient:	-
Flow Rate:	-
Solvent A:	-
Solvent B:	-
Chromatography Type:	Reversed phase

Chromatography ID:	CH003452
Chromatography Summary:	High pH (LC/MS Neg)
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	-
Flow Gradient:	-
Flow Rate:	-
Solvent A:	-
Solvent B:	-
Chromatography Type:	Reversed phase

Chromatography ID:	CH003453
Chromatography Summary:	HILIC (LC/MS Polar Neg)
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	-
Flow Gradient:	-
Flow Rate:	-
Solvent A:	-
Solvent B:	-
Chromatography Type:	HILIC

MS:

MS ID:	MS004336
Analysis ID:	AN004590
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolon (LC/MS Pos early)
Ion Mode:	POSITIVE

MS ID:	MS004337
Analysis ID:	AN004591
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolon (LC/MS Pos late)
Ion Mode:	POSITIVE

MS ID:	MS004338
Analysis ID:	AN004592
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolon (LC/MS Neg)
Ion Mode:	NEGATIVE

MS ID:	MS004339
Analysis ID:	AN004593
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolon (LC/MS Polar)
Ion Mode:	NEGATIVE