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MB Sample ID: SA213116
| Local Sample ID: | 625-1 |
| Subject ID: | SU002323 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002323 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 625-1 | SA213116 | FL025986 | Depdc5 KO | Genotype |
| 625-1 | SA213116 | FL025986 | Control | Treatment |
Collection:
| Collection ID: | CO002316 |
| Collection Summary: | Mouse cerebral cortex was rapidly dissected and flash frozen. |
| Sample Type: | Brain |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002335 |
| Treatment Summary: | Group housed mice, 6-9 weeks old Depdc5cc+ mice and their littermate controls, were randomized to fed or fasted conditions. Mice were weighed, followed by food removal at 1 pm for 24hr with ad lib water access. Twenty-four hours post-fasting, mice were weighed, followed by experimentation. |
Sample Preparation:
| Sampleprep ID: | SP002329 |
| Sampleprep Summary: | Mouse cortical lysate samples were rapidly isolated, and flash frozen in liquid nitrogen. Soluble metabolites were extracted directly from tissue using cold methanol/water (80/20, v/v) at approximately 1μL per 50μg of tissue, followed by ultrasonication (Branson Sonifier 250) for 15 s. Homogenized samples were incubated at −80 °C to precipitate proteins and subsequently centrifuged at 18,000xg for 20 min at 4 °C to pellet the debris. The supernatants containing soluble metabolites were collected in new tubes and evaporated to dryness using a SpeedVac concentrator (Thermo Savant). Next, the metabolites were reconstituted in acetonitrile/water (60/40, v/v), vortex-mixed, and centrifuged at 18,000xg for 30 min at 4°C to remove debris. |
Combined analysis:
| Analysis ID | AN003650 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Waters Xbridge Amide (100 x 3mm,3.5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH002704 |
| Chromatography Summary: | Samples were analyzed by High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry and Tandem Mass Spectrometry (HPLC-MS/MS). Specifically, system consisted of a Thermo Q-Exactive in line with an electrospray source and an Ultimate3000 (Thermo) series HPLC consisting of a binary pump, degasser, and auto-sampler outfitted with a Xbridge Amide column (Waters; dimensions of 3.0 mm × 100 mm and a 3.5 µm particle size). The mobile phase A contained 95% (vol/vol) water, 5% (vol/vol) acetonitrile, 10 mM ammonium hydroxide, 10 mM ammonium acetate, pH = 9.0; B was 100% Acetonitrile. The gradient was as following: 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A with a flow rate of 150 μL/min. The capillary of the ESI source was set to 275 °C, with sheath gas at 35 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, an m/z scan range from 60 to 900 was chosen and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 106 and the maximum injection time was 200 ms. The top 5 precursor ions were subsequently fragmented, in a data-dependent manner, using the higher energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500. Besides matching m/z, metabolites are identified by matching either retention time with analytical standards and/or MS2 fragmentation pattern. Data acquisition and analysis were carried out by Xcalibur 4.1 software and Tracefinder 4.1 software, respectively (both from Thermo Fisher Scientific). |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters Xbridge Amide (100 x 3mm,3.5um) |
| Flow Gradient: | 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A |
| Flow Rate: | 150 µL/min |
| Solvent A: | 95% water/5% acetonitrile; 10 mM ammonium hydroxide; 10 mM ammonium acetate, pH 9.0 |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003401 |
| Analysis ID: | AN003650 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The capillary of the ESI source was set to 275 °C, with sheath gas at 35 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, an m/z scan range from 60 to 900 was chosen and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 106 and the maximum injection time was 200 ms. The top 5 precursor ions were subsequently fragmented, in a data-dependent manner, using the higher energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500. Besides matching m/z, metabolites are identified by matching either retention time with analytical standards and/or MS2 fragmentation pattern. Data acquisition and analysis were carried out by Xcalibur 4.1 software and Tracefinder 4.1 software, respectively (both from Thermo Fisher Scientific). |
| Ion Mode: | UNSPECIFIED |
