Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA175294

Local Sample ID:	D2554
Subject ID:	SU001965
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001965
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
D2554	SA175294	FL021522	Donor	Group

Collection:

Collection ID:	CO001958
Collection Summary:	Cardiac tissue was excised and a mid-myocardial portion was used immediately for studies of mitochondrial respiration, or fixed in 4% paraformaldehyde (PFA) for paraffin embedding or in 4% PFA and 2% glutaraldehyde for TEM analysis. The remaining tissue was flash frozen in liquid nitrogen for all other assays.
Sample Type:	Heart

Treatment:

Treatment ID:	TR001977
Treatment Summary:	N/A

Sample Preparation:

Sampleprep ID:	SP001971
Sampleprep Summary:	Sample Preparation. Roughly 30 mg of frozen heart tissue were homogenized in 500 µl ice-cold methanol by bead beating (MP bioscience cat# 6913-100, Solon, OH) at 4°C (2 x 45 s). Metabolites and complex lipids were extracted using a biphasic separation with cold methyl tert-butyl ether (MTBE), methanol and water. Briefly, 1 ml of ice-cold MTBE was added to 300 μl of the homogenate spiked-in with 40 µl deuterated lipid internal standards (Sciex, cat#: 5040156, lot#: LPISTDKIT-101). The samples were then sonicated (3 x 30 s) and agitated at 4°C for 30 min. After addition of 250 μl of ice-cold water, the samples were vortexed for 1 min and centrifuged at 14,000 g for 5 min at 20°C. The upper organic phase contains the lipids, the lower aqueous phase contains the metabolites and the proteins are precipitated at the bottom of the tube. For quality controls, 3 reference plasma samples (40 µl plasma) and 1 preparation blank were processed in parallel. 1) Metabolites: Proteins were further precipitated by adding 700 μl of 33/33/33 acetone/acetonitrile/methanol spiked-in with 15 labeled metabolite internal standards to 300 μl of the aqueous phase and 200 μl of the lipid phase and incubating the samples overnight at -20°C. After centrifugation at 17,000 g for 10 min at 4°C, the metabolic extracts were dried down to completion and resuspended in 100 μl 50/50 methanol/water. 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 16,000 g for 5 min at 24°C.

Sampleprep ID:

SP001971

Sampleprep Summary:

Sample Preparation. Roughly 30 mg of frozen heart tissue were homogenized in 500 µl ice-cold methanol by bead beating (MP bioscience cat# 6913-100, Solon, OH) at 4°C (2 x 45 s). Metabolites and complex lipids were extracted using a biphasic separation with cold methyl tert-butyl ether (MTBE), methanol and water. Briefly, 1 ml of ice-cold MTBE was added to 300 μl of the homogenate spiked-in with 40 µl deuterated lipid internal standards (Sciex, cat#: 5040156, lot#: LPISTDKIT-101). The samples were then sonicated (3 x 30 s) and agitated at 4°C for 30 min. After addition of 250 μl of ice-cold water, the samples were vortexed for 1 min and centrifuged at 14,000 g for 5 min at 20°C. The upper organic phase contains the lipids, the lower aqueous phase contains the metabolites and the proteins are precipitated at the bottom of the tube. For quality controls, 3 reference plasma samples (40 µl plasma) and 1 preparation blank were processed in parallel. 1) Metabolites: Proteins were further precipitated by adding 700 μl of 33/33/33 acetone/acetonitrile/methanol spiked-in with 15 labeled metabolite internal standards to 300 μl of the aqueous phase and 200 μl of the lipid phase and incubating the samples overnight at -20°C. After centrifugation at 17,000 g for 10 min at 4°C, the metabolic extracts were dried down to completion and resuspended in 100 μl 50/50 methanol/water. 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 16,000 g for 5 min at 24°C.

Combined analysis:

Analysis ID	AN003055	AN003056
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS
Column	Thermo Accucore (150 x 2.1mm,2.6um)	Thermo Accucore (150 x 2.1mm,2.6um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	MS count (log2)	MS count (log2)

Chromatography:

Chromatography ID:	CH002262
Chromatography Summary:	Lipid extracts were also analyzed using an Ultimate 3000 RSLC system coupled with a Q Exactive mass spectrometer (Thermo Scientific, Waltham, MA) as previously described6. Each sample was run twice in positive and negative ionization modes. Lipids were separated using an Accucore C18 column 2.1 x 150 mm, 2.6 μm (Thermo Scientific) and mobile phase solvents consisted in 10 mM ammonium acetate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 10 mM ammonium acetate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The Q Exactive was equipped with a HESI-II prob
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Thermo Accucore (150 x 2.1mm,2.6um)
Solvent A:	60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium acetate
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002842
Analysis ID:	AN003055
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	LC-MS peak extraction, alignment, quantification and annotation was performed using LipidSearch software version 4.2 (Thermo Scientific). Only lipids present in >2/3 of the samples were kept for further analysis. Median normalization (excluding TAG and DAG) was applied to correct for differential starting material quantity. Missing values were imputed by drawing from a random distribution of low values in the corresponding sample. Lipids were identified by matching the precursor ion mass to a database and the experimental MS/MS spectra to a spectral library containing theoretical fragmentation spectra. The identity of cardiolipins, detected as [M-H]-, was manually validated by investigating individual MS/MS spectra. Lipid abundances were reported as spectral counts.
Ion Mode:	POSITIVE

MS ID:	MS002843
Analysis ID:	AN003056
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	LC-MS peak extraction, alignment, quantification and annotation was performed using LipidSearch software version 4.2 (Thermo Scientific). Only lipids present in >2/3 of the samples were kept for further analysis. Median normalization (excluding TAG and DAG) was applied to correct for differential starting material quantity. Missing values were imputed by drawing from a random distribution of low values in the corresponding sample. Lipids were identified by matching the precursor ion mass to a database and the experimental MS/MS spectra to a spectral library containing theoretical fragmentation spectra. The identity of cardiolipins, detected as [M-H]-, was manually validated by investigating individual MS/MS spectra. Lipid abundances were reported as spectral counts.
Ion Mode:	NEGATIVE