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MB Sample ID: SA353367
| Local Sample ID: | 30-08-18-2022-POS-Isaac-S07 |
| Subject ID: | SU003362 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003362 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 30-08-18-2022-POS-Isaac-S07 | SA353367 | FL041183 | Serum | Sample source |
| 30-08-18-2022-POS-Isaac-S07 | SA353367 | FL041183 | Liver-specific Gclc-Nrf2 DKO | Genotype |
Collection:
| Collection ID: | CO003355 |
| Collection Summary: | Mice were anesthetized with isoflurane, after which blood was collected via the retro-orbital venous sinus into BD microtainer tubes (BD #365967). Serum was isolated from the blood by centrifuging blood samples at 10000 xg for 5mins and stored at -80C. |
| Sample Type: | Blood (serum) |
Treatment:
| Treatment ID: | TR003371 |
| Treatment Summary: | WT (Gclc f/f), liver-specific Gclc KO (Gclc f/f), liver-specific Nrf2 KO (Nrf2 f/f) and liver-specific Gclc-Nrf2 DKO (Gclc f/f Nrf2 f/f) were induced by injecting mice via the tail vein with 2.5x1011 GC of AAV-TBG-Cre (Addgene, 107787-AAV8) in PBS. After 21 days, serum was isolated and stored at -80C. |
Sample Preparation:
| Sampleprep ID: | SP003369 |
| Sampleprep Summary: | For serum for WT, liver-specific Gclc KO, liver-specific Nrf2 KO, and liver-specific Gclc-Nrf2 DKO, 20 µL of mouse serum was combined with 180 µL of chloroform:methanol extraction solvent (v:v=1:2) containing internal standards at the final concentrations: 5nM D7-Sphinganine (Avanti Polar Lipids Inc., Cat# 860658), 12.5nM D3-Deoxysphinganine (Avanti Polar Lipids Inc., Cat# 860474), and SPLASH LIPIDOMIX (1:1000, Avanti Polar Lipids Inc., Cat# 330707). After sonicating (1400 rpm, 20°C, 5 min), the extracts were cleared by centrifugation (17,000g, 20°C, 10 min), and the lipids in the supernatant were analyzed by LC-MS. |
Combined analysis:
| Analysis ID | AN005313 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Perkin Elmer Brownlee SPP C18 (75 x 2.1mm, 2.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | Normalized to the median value of total lipid signals |
Chromatography:
| Chromatography ID: | CH004017 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Perkin Elmer Brownlee SPP C18 (75 x 2.1mm, 2.7um) |
| Column Temperature: | 50 |
| Flow Gradient: | 0–2 min 35% B, 2–8 min from 35 to 80% B, 8–22 min from 80 to 99% B, 22–36 min 99% B, and 36.1–40 min from 99 to 35% B |
| Flow Rate: | 0.400 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 10mM ammonium acetate |
| Solvent B: | 50% acetonitrile/50% isopropanol; 0.1% formic acid; 10mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005043 |
| Analysis ID: | AN005313 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS2 scan conditions were applied in positive mode, the scan range was from m/z 120–1000, resolution was 120,000 for MS, and 30,000 for DDMS2 (top 10), and the AGC target was 3E6 for full MS and 1E5 for DDMS2, allowing ions to accumulate for up to 200 ms for MS and 50 ms for MS/MS. For MS/MS, the following settings are used: isolation window width 1.2 m/z, stepped NCE at 10, 15, and 25 a.u., minimum AGC 5E2, and dynamic exclusion of previously sampled peaks for 8 s. Quality control (QC) samples were included to check the technical variability and were prepared by mixing an equal volume of lipid extract from each tissue or serum sample. QC samples were included in the analysis sequence every ten samples and monitored for changes in peak area, width, and retention time to determine the performance of the LC-MS/MS analysis. QC samples were subsequently used to align the analytical batches. The lipid peaks were identified, aligned, and exported using MS-DIAL. The data were further normalized to the median value of total lipid signals. Only lipids fully identified by MS2 spectra were included in the analysis. |
| Ion Mode: | POSITIVE |
