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MB Sample ID: SA175292
Local Sample ID: | D1234 |
Subject ID: | SU001965 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
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 |
---|---|---|---|---|
D1234 | SA175292 | 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. |
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 |