Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA319187

Local Sample ID:	CB040-V
Subject ID:	SU003050
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU003050
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
CB040-V	SA319187	FL037889	NA	type

Collection:

Collection ID:	CO003043
Collection Summary:	Plasma was collected from umbilical cords at the Lucile Packard Children’s Hospital Stanford with permission from the Institutional Review Board (IRB #46411). All umbilical cords were collected and sent for clinically-indicated testing to Pathology where blood gas analysis was performed following Stanford protocol. Leftover and discarded samples were obtained for research. Arterial and venous blood were extracted from umbilical cords by heparinized syringe and transferred to low binding tubes.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR003059
Treatment Summary:	Samples were centrifuged at 1300g for 10 minutes. Plasma was aliquoted in cryovials and frozen at -80°C.

Sample Preparation:

Sampleprep ID:	SP003056
Sampleprep Summary:	Metabolites and lipids were extracted in 96-well high throughput fashion using a liquid-liquid biphasic separation with cold methyl tert-butyl ether (MTBE), methanol, and water. To begin, 1 mL MTBE was added to 40 μl of plasma and spiked with 40 μl of deuterated lipid internal standards (Sciex, cat# 5040156, lot# LPISTDKIT-103). The samples were agitated at 4°C for 30 minutes. After the addition of 250 μl cold water, samples were vortexed for 1 minute then centrifuged at 3,800 g for 5 minutes at 4°C. The upper organic phase contained the lipids while the lower aqueous phase contained metabolites with precipitated proteins at the bottom of the tube. For quality control, reference plasma samples (40 μl plasma), as well as controls lacking samples (blanks), were processed in parallel. 1) Metabolites: To further precipitate proteins, 500 μl 1:1:1 acetone: acetonitrile: methanol spiked with 16 labeled metabolite internal standards was added to 300 μl of the aqueous phase and 200 μl of the organic phase and incubated overnight at -20°C. After centrifugation at 3,800 g for 10 min at 4°C, the metabolic extracts were dried down under a stream of nitrogen gas and resuspended in 100 μl 50/50 methanol/water for LC-MS. 2) Complex lipids: 700 µl of the organic phase was dried down under a stream of nitrogen and resolubilized in 200 μl of methanol for storage at -20°C until analysis. The day of the analysis, samples were dried down, resuspended in 300 μl of 10 mM ammonium acetate in 90/10 methanol/toluene, and centrifuged at 3,800 g for 5 min at 4°C.

Sampleprep ID:

SP003056

Sampleprep Summary:

Metabolites and lipids were extracted in 96-well high throughput fashion using a liquid-liquid biphasic separation with cold methyl tert-butyl ether (MTBE), methanol, and water. To begin, 1 mL MTBE was added to 40 μl of plasma and spiked with 40 μl of deuterated lipid internal standards (Sciex, cat# 5040156, lot# LPISTDKIT-103). The samples were agitated at 4°C for 30 minutes. After the addition of 250 μl cold water, samples were vortexed for 1 minute then centrifuged at 3,800 g for 5 minutes at 4°C. The upper organic phase contained the lipids while the lower aqueous phase contained metabolites with precipitated proteins at the bottom of the tube. For quality control, reference plasma samples (40 μl plasma), as well as controls lacking samples (blanks), were processed in parallel. 1) Metabolites: To further precipitate proteins, 500 μl 1:1:1 acetone: acetonitrile: methanol spiked with 16 labeled metabolite internal standards was added to 300 μl of the aqueous phase and 200 μl of the organic phase and incubated overnight at -20°C. After centrifugation at 3,800 g for 10 min at 4°C, the metabolic extracts were dried down under a stream of nitrogen gas and resuspended in 100 μl 50/50 methanol/water for LC-MS. 2) Complex lipids: 700 µl of the organic phase was dried down under a stream of nitrogen and resolubilized in 200 μl of methanol for storage at -20°C until analysis. The day of the analysis, samples were dried down, resuspended in 300 μl of 10 mM ammonium acetate in 90/10 methanol/toluene, and centrifuged at 3,800 g for 5 min at 4°C.

Combined analysis:

Analysis ID	AN004817	AN004818	AN004819	AN004820	AN004821	AN004822
Analysis type	MS	MS	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC	None (Direct infusion)	None (Direct infusion)
Chromatography system	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000	Thermo Vanquish	Thermo Vanquish	Shimazdu LC-30AD	Shimazdu LC-30AD
Column	Agilent Zorbax SBaq (50 x 2.1mm x 1.7 um)	Agilent Zorbax SBaq (50 x 2.1mm x 1.7 um)	Merck SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)	Merck SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)	None	None
MS Type	ESI	ESI	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap	QTRAP	QTRAP
MS instrument name	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	ABI Sciex 5500 QTrap	ABI Sciex 5500 QTrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Relative Abundance	Relative Abundance	Relative Abundance	Relative Abundance	Relative Abundance	Relative Abundance

Chromatography:

Chromatography ID:	CH003640
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Agilent Zorbax SBaq (50 x 2.1mm x 1.7 um)
Column Temperature:	60
Flow Gradient:	N/A
Flow Rate:	0.6 ml/min
Solvent A:	0.06% acetic acid in water
Solvent B:	0.06% acetic acid in methanol
Chromatography Type:	Reversed phase

Chromatography ID:	CH003641
Instrument Name:	Thermo Vanquish
Column Name:	Merck SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:	40
Flow Gradient:	N/A
Flow Rate:	0.5 ml/min
Solvent A:	10 mM ammonium acetate in 50/50 acetonitrile/water
Solvent B:	10 mM ammonium acetate in 95/5 acetonitrile/water
Chromatography Type:	HILIC

Chromatography ID:	CH003642
Instrument Name:	Shimazdu LC-30AD
Column Name:	None
Column Temperature:	20
Flow Gradient:	N/A
Flow Rate:	0.15 ml/min
Solvent A:	9:1 Methanol Toluene with 10 mM ammonium acetate
Solvent B:	N/A
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS004563
Analysis ID:	AN004817
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data from each mode were independently analyzed using Progenesis QI software (v2.3) (Nonlinear Dynamics, Durham, NC). Metabolic features from blanks and those that did not show sufficient linearity upon dilution in QC samples (r<0.6) were discarded. Only metabolic features present in >2/3 of the samples were kept for further analysis. Missing values were imputed by drawing from a random distribution of low values in the corresponding sample. Intensity drift was corrected using SERRF. Data quality post-normalization was verified by ensuring clustering of pooled sample replicates on a principal component analysis (PCA) plot.
Ion Mode:	POSITIVE

MS ID:	MS004564
Analysis ID:	AN004818
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data from each mode were independently analyzed using Progenesis QI software (v2.3) (Nonlinear Dynamics, Durham, NC). Metabolic features from blanks and those that did not show sufficient linearity upon dilution in QC samples (r<0.6) were discarded. Only metabolic features present in >2/3 of the samples were kept for further analysis. Missing values were imputed by drawing from a random distribution of low values in the corresponding sample. Intensity drift was corrected using SERRF. Data quality post-normalization was verified by ensuring clustering of pooled sample replicates on a principal component analysis (PCA) plot.
Ion Mode:	NEGATIVE

MS ID:	MS004565
Analysis ID:	AN004819
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data from each mode were independently analyzed using Progenesis QI software (v2.3) (Nonlinear Dynamics, Durham, NC). Metabolic features from blanks and those that did not show sufficient linearity upon dilution in QC samples (r<0.6) were discarded. Only metabolic features present in >2/3 of the samples were kept for further analysis. Missing values were imputed by drawing from a random distribution of low values in the corresponding sample. Intensity drift was corrected using SERRF. Data quality post-normalization was verified by ensuring clustering of pooled sample replicates on a principal component analysis (PCA) plot.
Ion Mode:	POSITIVE

MS ID:	MS004566
Analysis ID:	AN004820
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data from each mode were independently analyzed using Progenesis QI software (v2.3) (Nonlinear Dynamics, Durham, NC). Metabolic features from blanks and those that did not show sufficient linearity upon dilution in QC samples (r<0.6) were discarded. Only metabolic features present in >2/3 of the samples were kept for further analysis. Missing values were imputed by drawing from a random distribution of low values in the corresponding sample. Intensity drift was corrected using SERRF. Data quality post-normalization was verified by ensuring clustering of pooled sample replicates on a principal component analysis (PCA) plot.
Ion Mode:	NEGATIVE

MS ID:	MS004567
Analysis ID:	AN004821
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	Lipidyzer data were reported by the Lipidomics Workflow Manager (LWM, v1.0.5.0) software which calculates concentrations for each detected lipid as average intensity of the analyte MRM relative to the average intensity of the most structurally similar internal standard (IS) MRM multiplied by its concentration. Lipids detected in less than 2/3 of the samples were discarded and missing values were imputed by drawing from a random distribution of low values class-wise in the corresponding sample. Data quality was verified by ensuring clustering of the quality control replicates analyzed on a PCA plot. We detected lipid species belonging to 13 classes (e.g. CE, CER, DAG, FFA, HCER, LCER, DCER, LPE, LPC, PC, PE, SM, TAG) and their abundance were reported as concentrations in nmol/g. The Q1 and Q3 mass provided in the metadata were used to target specific lipid classes. Lipids with the designated Q1 mass, also known as the parent ion, are selected from the first quadrupole. The lipids are then fragmented and sent to the third quadrupole. In the third quadrupole, the desired lipid class is selected based on its Q3 mass.
Ion Mode:	POSITIVE

MS ID:	MS004568
Analysis ID:	AN004822
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	Lipidyzer data were reported by the Lipidomics Workflow Manager (LWM, v1.0.5.0) software which calculates concentrations for each detected lipid as average intensity of the analyte MRM relative to the average intensity of the most structurally similar internal standard (IS) MRM multiplied by its concentration. Lipids detected in less than 2/3 of the samples were discarded and missing values were imputed by drawing from a random distribution of low values class-wise in the corresponding sample. Data quality was verified by ensuring clustering of the quality control replicates analyzed on a PCA plot. We detected lipid species belonging to 13 classes (e.g. CE, CER, DAG, FFA, HCER, LCER, DCER, LPE, LPC, PC, PE, SM, TAG) and their abundance were reported as concentrations in nmol/g. The Q1 and Q3 mass provided in the metadata were used to target specific lipid classes. Lipids with the designated Q1 mass, also known as the parent ion, are selected from the first quadrupole. The lipids are then fragmented and sent to the third quadrupole. In the third quadrupole, the desired lipid class is selected based on its Q3 mass.
Ion Mode:	NEGATIVE