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MB Sample ID: SA082253
| Local Sample ID: | F3 |
| Subject ID: | SU001251 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001251 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| F3 | SA082253 | FL012477 | NASH Liver Tissue from Patient | Source |
Collection:
| Collection ID: | CO001245 |
| Collection Summary: | De-identified tissues were obtained from Magee Women’s Hospital (Pittsburgh, PA) and the University of Washington Department of Pediatrics, Division of Genetic Medicine, Laboratory of Developmental Biology (Seattle, WA) after obtaining a written informed consent by a protocol approved by the Human Research Review Committee of the University of Pittsburgh (Honest broker approval number HB015 and HB000836). Human fetal liver tissue and/or cells were isolated and culture from fetal livers as previously described (26890260). The de-identified normal human liver tissue and/or cells were obtained through the Liver Tissue Cell Distribution System (Pittsburgh, PA) after obtaining a written informed consent by a protocol approved by the Human Research Review Committee of the University of Pittsburgh, which was funded by NIH Contract # HSN276201200017C. Adult human liver tissue and/or cells were also obtained from Ira J Fox Laboratory at Children’s Hospital of UPMC, after obtaining a written informed consent by a protocol approved by the Human Research Review Committee and the Institutional Review Board (IRB#: PRO12090466) of the University of Pittsburgh. |
| Sample Type: | Liver |
Treatment:
| Treatment ID: | TR001266 |
| Treatment Summary: | Normal liver tissue and NASH liver tissue samples were not treated, and were directly analyzed for lipidomics profile. Bioengineered human iPS-derive fatty liver tissue was synthesized as described in the study referenced herein |
Sample Preparation:
| Sampleprep ID: | SP001259 |
| Sampleprep Summary: | Human normal liver, human NASH liver and human iPS-derived fatty liver tissueiKD-SIRT1 samples were homogenized using a FastPrep system (MP Bio) with Matrix D ceramic beads in 80% MeOH at a ratio of 15 μL/mg tissue. The homogenate was spiked with isotopically labelled standards, taurine-1,1,2,2-d4 (final concentration 100 μM, Cambridge Isotopes MA) and 10 μL of a 50 μg/mL fatty acid internal standard mix. Chloroform (600 μL) was then added to the homogenate supernatant and the sample was vortexed and centrifuged at 1,500 x g for 5 min. The aqueous phase was taken for polar metabolite analysis and the organic phase was split for targeted free fatty acid analysis and untargeted lipidomics. Polar samples were cleared by centrifugation at 16,000 x g and the supernatant dried under N2. Samples were resuspended in 50 μL of 1.5 mM ammonium fluoride (aq) and 10 μL was injected for separation and analysis. The organic phase was dried under N2 and reconstituted in 100 μL of chloroform:methanol (2:1) and 5 μL was injected for untargeted lipidomics analysis. |
Combined analysis:
| Analysis ID | AN001967 | AN001968 | AN001969 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish |
| Column | Thermo Fisher Accucore C18 (100 x 2.1mm,5um) | Thermo Fisher Accucore C18 (100 x 2.1mm,5um) | Phenomenex Kinetex C18+ (100 x 2.1mm,1.7um) |
| MS Type | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | UNSPECIFIED |
| Units | Peak area normalized to internal standard | Peak area normalized to internal standard | Peak Area |
Chromatography:
| Chromatography ID: | CH001423 |
| Chromatography Summary: | LIPIDOMICS: Samples were separated on a Thermo Fisher Accucore C18 column (2.1 X 100 mm, 5 μ pore size) using solvent A (H2O:ACN (1:1) with 10 mM ammonium acetate + 0.1% formic acid) and solvent B (IPA:ACN (9:1) with 10 mM ammonium acetate + 0.1% formic acid) at a flow rate of 0.2 mL/min. The gradient started at 0%B and increased to 50%B from 2-10 min following a second increase to 95%B from 10-47 min. The gradient was held for 4 min at 95%B before increasing to 100%B at 51 min for a 6 min wash. At 57 min the system was returned to initial conditions to equilibrate before the next injection. Total run time was 60 min. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Fisher Accucore C18 (100 x 2.1mm,5um) |
| Flow Gradient: | The gradient started at 0%B and increased to 50%B from 2-10 min following a second increase to 95%B from 10-47 min. The gradient was held for 4 min at 95%B before increasing to 100%B at 51 min for a 6 min wash. At 57 min the system was returned to initial conditions to equilibrate before the next injection. Total run time was 60 min. |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 50% water/50% acetonitrile; 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 |
| Chromatography ID: | CH001424 |
| Chromatography Summary: | METABOLOMICS: Briefly, samples were separated over a reversed phase Phenomenex Kinetex C18+ column (2.1 × 100 mm, 1.7 μm particle size) maintained at 40°C. For the 20 minute LC gradient, the mobile phase consisted of the following: solvent A (1.5mM ammonium fluoride) and solvent B (100% acetonitrile). The gradient was the following: 0-12.0 min 5% B, to 100% B, 12.0-15.0 min hold at 100% B, 15.0-15.1 100% to 5% B, 15.1-20.0 min 5%B. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18+ (100 x 2.1mm,1.7um) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001821 |
| Analysis ID: | AN001967 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | LIPIDOMICS: Samples were analyzed using full scan accurate mass at a resolution of 70K in positive and negative mode and 17.5K for ddMS2. Thermo Fisher LipidSearch 4.2.2 software was used for peak quantification, alignment, and MS2 identification. Peak areas were normalized to internal standard. |
| Ion Mode: | POSITIVE |
| MS ID: | MS001822 |
| Analysis ID: | AN001968 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | LIPIDOMICS: Samples were analyzed using full scan accurate mass at a resolution of 70K in positive and negative mode and 17.5K for ddMS2. Thermo Fisher LipidSearch 4.2.2 software was used for peak quantification, alignment, and MS2 identification. Peak areas were normalized to internal standard. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS001823 |
| Analysis ID: | AN001969 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | METABOLOMICS: Metabolites were identified in both positive and negative mode by accurate mass (≤ 10 ppm) and retention time. Integrated peak areas were then extracted manually using Quan Browser (Thermo Fisher Xcalibur ver 2.7) and normalized to internal standard. |
| Ion Mode: | UNSPECIFIED |
