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MB Sample ID: SA212523
| Local Sample ID: | 37908 _P |
| Subject ID: | SU002315 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL6 and FVB mixed strain |
| Age Or Age Range: | 54-59 weeks old |
| Gender: | Male and female |
| Animal Feed: | Specialty Feeds Irradiated Rat and Mouse Standard Chow Diet |
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Subject:
| Subject ID: | SU002315 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL6 and FVB mixed strain |
| Age Or Age Range: | 54-59 weeks old |
| Gender: | Male and female |
| Animal Feed: | Specialty Feeds Irradiated Rat and Mouse Standard Chow Diet |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 37908 _P | SA212523 | FL025929 | Male | Sex |
| 37908 _P | SA212523 | FL025929 | ERalpha-knockout | Genotype |
Collection:
| Collection ID: | CO002308 |
| Collection Summary: | Ventricles were dissected from mice such that each sample will include left and right ventricular tissue.Tissues were snap frozen in liquid nitrogen and stored in -80 freezer until tissues were processed for lipid extractions Blood was collected via cardiac puncture and stored in EDTA tubes on ice. EDTA tubes were spun at 3000g for 15 mins at 4 degrees. Plasma was then collected from these tubes (supernatant) and then stored at -80 degrees. |
| Sample Type: | Ventricles, Plasma, Skeletal Muscles, Liver, Epididymal fat |
| Storage Conditions: | -80℃ |
| Collection Vials: | 1.5ml eppendorf tubes |
| Storage Vials: | 1.5ml eppendorf tubes |
Treatment:
| Treatment ID: | TR002327 |
| Treatment Summary: | Mice did not undergo specific treatment, as this was a basal phenotyping study. Mice were fasted for 6 hours before dissections, and a lethal dose of anesthesia was delivered via intraperitoneal injection before tissue collection. |
| Animal Anesthesia: | Pentobarbitone |
| Animal Fasting: | 6 hours |
Sample Preparation:
| Sampleprep ID: | SP002321 |
| Sampleprep Summary: | tissues was homogenised in 1xPBS and then sonicated with a probe-sonicator for 15 seconds, 23 amplitude. BCA assays were then conducted to determine protein concentrations of these homogenates. Lipid extraction was conducted using 10ul of sample (ventricle, skeletal muscle homogenate at 5mg/ml, fat homogenate at 2mg/ml and liver homogenate at 2.5mg/ml) using the single phase chloroform methanol method. 10ul of internal standards and 200ul of chloroform:methanol (1:2) were added to samples before the mixture was vortexed. Samples were then placed on a rotary shaker for 10 mins at a speed of 90 before being transferred to a bath sonicator. Samples were then sonicated for 30 mins at water temperature below 28 degrees. Samples were then removed and rested at room temperature for 20 mins. Samples were then centrifuged at 13000rpm for 10 minutes. 200ul of the supernatant was then transferred to 0.5ml polypropylene 96 well plates, and spun dried using a speedvac vacuum concentrator. Lipids were reconstituted in 50ul water saturated butanol + 50ul of Ammonium Formate. |
| Sampleprep Protocol Filename: | Agilent_appnote |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003638 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Agilent 1290 Infinity II |
| Column | Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Agilent 6490 QQQ |
| Ion Mode | POSITIVE |
| Units | pmol per mg (tissues) pmol per ml (plasma) |
Chromatography:
| Chromatography ID: | CH002693 |
| Chromatography Summary: | The running solvent consisted of solvent A: 50% H2O / 30% acetonitrile / 20% isopropanol (v/v/v) containing 10mM ammonium formate and 5uM medronic acid, and solvent B: 1% H2O / 9% acetonitrile / 90% isopropanol (v/v/v) containing 10mM ammonium formate. We utilized a stepped linear gradient with a 16-minute cycle time per sample and a 1µL sample injection. To increase throughput, we used a dual column set up to equilibrate the second column while the first is running a sample. The sample analytical gradient was as follows: starting with a flow rate of 0.4mL/minute at 15% B and increasing to 50% B over 2.5 minutes, then to 57% over 0.1 minutes, to 70% over 6.4 minutes, to 93% over 0.1 minute, to 96% over 1.9 minutes and finally to 100% over 0.1 minute. The solvent was then held at 100% B for 0.9 minutes (total 12.0 minutes). Equilibration was started as follows: solvent was decreased from 100% B to 15% B over 0.2 minutes and held until a total of 16 minutes. The next sample is injected and the columns are switched. |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) |
| Flow Gradient: | starting with a flow rate of 0.4mL/minute at 15% B and increasing to 50% B over 2.5 minutes, then to 57% over 0.1 minutes, to 70% over 6.4 minutes, to 93% over 0.1 minute, to 96% over 1.9 minutes and finally to 100% over 0.1 minute. The solvent was then held at 100% B for 0.9 minutes (total 12.0 minutes). Equilibration was started as follows: solvent was decreased from 100% B to 15% B over 0.2 minutes and held until a total of 16 minutes. |
| Flow Rate: | 0.4mL/min |
| Solvent A: | 50% water/30% acetonitrile/20% isopropanol; 10mM ammonium formate; 5uM medronic acid |
| Solvent B: | 1% water/9% acetonitrile/90% isopropanol; 10mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003389 |
| Analysis ID: | AN003638 |
| Instrument Name: | Agilent 6490 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Details previously published in https://doi.org/10.1016/j.chembiol.2018.10.008 Analysis of plasma extracts was performed on an Agilent 6490 QQQ mass spectrometer with an Agilent 1290 series HPLC system and a ZORBAX eclipse plus C18 column (2.1x100mm 1.8μm, Agilent) with the thermostat set at 60°C. Mass spectrometry analysis was performed in positive ion mode with dynamic scheduled multiple reaction monitoring (MRM). Mass spectrometry settings and MRM transitions for each lipid class, subclass and individual species are shown in Tables 1 and S1. The solvent system consisted of solvent A) 50% H2O / 30% acetonitrile / 20% isopropanol (v/v/v) containing 10mM ammonium formate and solvent B) 1% H2O / 9% acetonitrile / 90% isopropanol (v/v/v) containing 10mM ammonium formate. We utilized a stepped linear gradient with a 15-minute cycle time per sample and a 1μL sample injection. The gradient was as follows; starting with a flow rate of 0.4ml/minute at 10% B and increasing to 45% B over 2.7 minutes, then to 53% over 0.1 minutes, to 65% over 6.2 minutes, to 89% over 0.1 minute, to 92% over 1.9 minutes and finally to 100% over 0.1 minute. The solvent was then held at 100% B for 0.8 minutes (total 11.9 minutes). Equilibration was as follows, solvent was decreased from 100% B to 10% B over 0.1 minute and held for an additional 0.9 minutes. Flow rate was then switched to 0.6 ml/minute for 1 minute before returning to 0.4 ml/minute over 0.1 minutes. Solvent B was held at 10% B for a further 0.9 minutes at 0.4ml/minutes for a total cycle time of 15 minutes. The following mass spectrometer conditions were used; gas temperature, 150°C, gas flow rate 17L/min, nebulizer 20psi, Sheath gas temperature 200°C, capillary voltage 3500V and sheath gas flow 10L/min. Isolation widths for Q1 and Q3 were set to “unit” resolution (0.7 amu). PQC samples consisting of a pooled set of 6 healthy individuals were incorporated into the analysis at 1 PQC per 18 plasma samples. TQC consisted of PQC extracts which were pooled and split into individual vials to provide a measure of technical variation from the mass spectrometer only. These were included at a ratio of 1 TQC per 18 plasma samples. TQCs were monitored for changes in peak area, width and retention time to determine the performance of the LC-MS/MS analysis and were subsequently used to align for differential responses across the analytical batches. |
| Ion Mode: | POSITIVE |
