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MB Sample ID: SA289470
| Local Sample ID: | ANP_experiment_set_3_replicate_3 |
| Subject ID: | SU002858 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002858 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| ANP_experiment_set_3_replicate_3 | SA289470 | FL035708 | 2 μl | Factor |
Collection:
| Collection ID: | CO002851 |
| Collection Summary: | Mouse metabolites were collected from the liver of female mice using methanol extraction. After euthanizing a mouse, the liver was immediately frozen in liquid nitrogen. We then used cold (-20C or colder) 80% methanol to extract metabolites. First, 1 ml of 80% methanol was added to the liver and incubated for 10 min at -20oC. Glass beads were added to the liver and then the liver was lysed by bead-beating for 45 s using a Tissuelyser cell disrupter (Qiagen). The lysate was incubated for 10 min at -20oC and centrifuged (13200 rpm, 5 min) to separate metabolites from macromolecules. The supernatant was collected and 200 µl of 80% methanol was added to the pellet. The incubation, shaking and centrifugation steps were repeated twice to extract more metabolites from the pellet. The three supernatants were combined and centrifuged (14000 rpm, 10 min) to separate any remaining macromolecules from the metabolites. The combined supernatants were dried using a SpeedVac Concentrator (Savant, SPD131DDA) at 25oC and the dried metabolite samples were stored at -80oC. |
| Sample Type: | Liver |
| Collection Method: | 80% methanol |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002867 |
| Treatment Summary: | All samples were treated identically for this sub-study, except a higher volume (4 μl ANP_experiment_set_3_replicate_1) of one sample was injected than for the other samples (2 μl). |
Sample Preparation:
| Sampleprep ID: | SP002864 |
| Sampleprep Summary: | On the day of metabolite analysis, dried-down extracts were reconstituted in 150 µl 70% acetonitrile, at a relative protein concentration of ~ 2 µg/µl. Either 4 µl (replicate 1) or 2 µl (all other replicates) of this reconstituted extract was injected for LC/MS-based targeted and untargeted metabolite profiling. |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN004463 | AN004464 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Normal phase | Normal phase |
| Chromatography system | Agilent Model 1290 Infinity II liquid chromatography system | Agilent Model 1290 Infinity II liquid chromatography system |
| Column | Cogent Diamond Hydride (150 × 2.1 mm, 4um) | Cogent Diamond Hydride (150 × 2.1 mm, 4um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Agilent 6550 QTOF | Agilent 6550 QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Ion abundance (peak area) | Ion abundance (peak area) |
Chromatography:
| Chromatography ID: | CH003350 |
| Chromatography Summary: | Tissue extracts were analyzed by LC/MS as described previously, using a platform comprised of an Agilent Model 1290 Infinity II liquid chromatography system coupled to an Agilent 6550 iFunnel time-of-flight MS analyzer. Chromatography of metabolites utilized aqueous normal phase (ANP) chromatography on a Diamond Hydride column (Microsolv). Mobile phases consisted of: (A) 50% isopropanol, containing 0.025% acetic acid, and (B) 90% acetonitrile containing 5 mM ammonium acetate. To eliminate the interference of metal ions on chromatographic peak integrity and electrospray ionization, EDTA was added to the mobile phase at a final concentration of 5 µM. The following gradient was applied: 0-1.0 min, 99% B; 1.0-15.0 min, to 20% B; 15.0 to 29.0, 0% B; 29.1 to 37min, 99% B. |
| Instrument Name: | Agilent Model 1290 Infinity II liquid chromatography system |
| Column Name: | Cogent Diamond Hydride (150 × 2.1 mm, 4um) |
| Column Temperature: | 26 |
| Flow Gradient: | The following gradient was applied: 0-1.0 min, 99% B; 1.0-15.0 min, to 20% B; 15.0 to 29.0, 0% B; 29.1 to 37min, 99% B. |
| Flow Rate: | 0.3 mL/min |
| Solvent A: | 50% isopropanol/50% water; 0.025% acetic acid |
| Solvent B: | 90% acetonitrile/10% water; 5 mM ammonium acetate |
| Chromatography Type: | Normal phase |
MS:
| MS ID: | MS004210 |
| Analysis ID: | AN004463 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | LC/MS-based targeted and untargeted metabolite profiling. For targeted analysis, raw LC/MS data was extracted by MassProfinder 8.0 (Agilent Technologies) using an in-house annotated personal metabolite database that contains 863 metabolites (Agilent Technologies). Additionally, molecular feature extraction (MFE) was performed for untargeted metabolite profiling using MassProfinder 8.0 (Agilent Technologies). The untargeted molecular features were imported into MassProfiler Professional 15.1 (MPP, Agilent Technologies) and searched against Metlin personal metabolite database (PCDL database 8.0), Human Metabolome Database (HMDB) and an in-house phospholipid database for tentative metabolite ID assignments, based on monoisotopic neutral mass (< 5 ppm mass accuracy) matches. Furthermore, a molecular formula generator (MFG) algorithm in MPP was used to generate and score empirical molecular formulae, based on a weighted consideration of monoisotopic mass accuracy, isotope abundance ratios, and spacing between isotope peaks. A tentative compound ID was assigned when PCDL database and MFG scores concurred for a given candidate molecule. Tentatively assigned molecules were reextracted using Profinder 8.0 for confirmation of untargeted results. Only non-lipid metabolites that were identified in study ST002349 were retained for this analysis. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004211 |
| Analysis ID: | AN004464 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | LC/MS-based targeted and untargeted metabolite profiling. For targeted analysis, raw LC/MS data was extracted by MassProfinder 8.0 (Agilent Technologies) using an in-house annotated personal metabolite database that contains 863 metabolites (Agilent Technologies). Additionally, molecular feature extraction (MFE) was performed for untargeted metabolite profiling using MassProfinder 8.0 (Agilent Technologies). The untargeted molecular features were imported into MassProfiler Professional 15.1 (MPP, Agilent Technologies) and searched against Metlin personal metabolite database (PCDL database 8.0), Human Metabolome Database (HMDB) and an in-house phospholipid database for tentative metabolite ID assignments, based on monoisotopic neutral mass (< 5 ppm mass accuracy) matches. Furthermore, a molecular formula generator (MFG) algorithm in MPP was used to generate and score empirical molecular formulae, based on a weighted consideration of monoisotopic mass accuracy, isotope abundance ratios, and spacing between isotope peaks. A tentative compound ID was assigned when PCDL database and MFG scores concurred for a given candidate molecule. Tentatively assigned molecules were reextracted using Profinder 8.0 for confirmation of untargeted results. Only non-lipid metabolites that were identified in study ST002349 were retained for this analysis. |
| Ion Mode: | NEGATIVE |
