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MB Sample ID: SA175276
Local Sample ID: | M2800 |
Subject ID: | SU001964 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|---|
M2800 | SA175276 | 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. |
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 |