Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA175281

Local Sample ID:	M2856
Subject ID:	SU001964
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001964
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
M2856	SA175281	FL021519	Hypertophic cardiomyopathy	Group

Collection:

Collection ID:	CO001957
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:	TR001976
Treatment Summary:	N/A

Sample Preparation:

Sampleprep ID:	SP001970
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:

SP001970

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	AN003051	AN003052	AN003053	AN003054
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS
Column	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)	Agilent Zorbax SBaq (50 x 2.1mm,1.7um)	Agilent Zorbax SBaq (50 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	MS count (log2)	MS count (log2)	MS count (log2)	MS count (log2)

Chromatography:

Chromatography ID:	CH002260
Chromatography Summary:	HILIC experiments were performed using a ZIC-HILIC column 2.1 x 100 mm, 3.5 μm, 200Å (Merck Millipore, Darmstadt, Germany) and mobile phase solvents consisting of 10 mM ammonium acetate in 50/50 acetonitrile/water (A) and 10 mM ammonium acetate in 95/5 acetonitrile/water (B).
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Solvent A:	50% acetonitrile/50% water; 10 mM ammonium acetate
Solvent B:	95% acetonitrile/5% water; 10 mM ammonium acetate
Chromatography Type:	HILIC

Chromatography ID:	CH002261
Chromatography Summary:	RPLC experiments were performed using a Zorbax SBaq column 2.1 x 50 mm, 1.7 μm, 100Å (Agilent Technologies, Palo Alto, CA) and mobile phase solvents consisting of 0.06% acetic acid in water (A) and 0.06% acetic acid in methanol (B).
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Agilent Zorbax SBaq (50 x 2.1mm,1.7um)
Solvent A:	100% water; 0.06% acetic acid
Solvent B:	100% methanol; 0.06% acetic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002838
Analysis ID:	AN003051
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 didn’t 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. Median normalization 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. Data from each mode were merged and metabolites of interest were formally identified by matching fragmentation spectra and retention time to analytical-grade standards when possible or matching experimental MS/MS to fragmentation spectra in publicly available databases.
Ion Mode:	POSITIVE

MS ID:	MS002839
Analysis ID:	AN003052
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 didn’t 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. Median normalization 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. Data from each mode were merged and metabolites of interest were formally identified by matching fragmentation spectra and retention time to analytical-grade standards when possible or matching experimental MS/MS to fragmentation spectra in publicly available databases.
Ion Mode:	NEGATIVE

MS ID:	MS002840
Analysis ID:	AN003053
Instrument Name:	Thermo Q Exactive 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 didn’t 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. Median normalization 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. Data from each mode were merged and metabolites of interest were formally identified by matching fragmentation spectra and retention time to analytical-grade standards when possible or matching experimental MS/MS to fragmentation spectra in publicly available databases.
Ion Mode:	POSITIVE

MS ID:	MS002841
Analysis ID:	AN003054
Instrument Name:	Thermo Q Exactive 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 didn’t 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. Median normalization 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. Data from each mode were merged and metabolites of interest were formally identified by matching fragmentation spectra and retention time to analytical-grade standards when possible or matching experimental MS/MS to fragmentation spectra in publicly available databases.
Ion Mode:	NEGATIVE