Return to study ST002056 main page
MB Sample ID: SA193793
| Local Sample ID: | CarnsTG2 |
| Subject ID: | SU002138 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Age Or Age Range: | 8-12 weeks |
| Gender: | Male |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002138 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Age Or Age Range: | 8-12 weeks |
| Gender: | Male |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CarnsTG2 | SA193793 | FL023736 | CarnsTG | Genotype |
Collection:
| Collection ID: | CO002131 |
| Collection Summary: | To determine the effects of the Carns overexpression on the global cardio metabolomic profile under the basal conditions, hearts were collected from the WT and CarnsTg mice upon sacrifice and snap-frozen in liquid nitrogen. |
| Collection Protocol ID: | 18404 |
| Collection Protocol Filename: | Metabolomics |
| Collection Protocol Comments: | Effect of Carns overexpression |
| Sample Type: | Heart |
| Collection Location: | University of Louisville |
| Storage Conditions: | -80℃ |
| Collection Tube Temp: | -80 |
Treatment:
| Treatment ID: | TR002150 |
| Treatment Summary: | To determine the changes in the cardio metabolomic profile associated with the ischemic injury and whether Carns overexpression influences the cardiac metabolism, isolated hearts from the WT and CarnsTg mice hearts were perfused in the Langendorff mode for 35 min, perfused for 20 min followed by 5 min of ischemia and perfused for 20 min followed by 15 min of ischemia as described previousl |
Sample Preparation:
| Sampleprep ID: | SP002144 |
| Sampleprep Summary: | To extract polar metabolites, 800 µL of methanol was added to 200 µL of homogenized heart sample. Mixture was vortexed for 2 min and then placed on ice for 10 min, followed by another 2 min of vortex mixing, and centrifuged at 15,000 rpm for 20 min at 4 °C. Supernatant was transferred into a glass vial and dried in a SpeedVac evaporator to remove methanol, followed by lyophilization to remove water. The dried metabolite extract was dissolved with 30 µL of 20 mg/mL methoxyamine hydrochloride pyridine solution followed by vigorous vortex mixing for 1 min. Methoxylation was performed by sonicating the sample for 20 min and incubation at 60 °C for 1 h. Derivatization was performed by adding 20 µL of N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) or N-Methyl-N-tert-butyldimethysilyltrifuoroacetamide (MTBSTFA) with 1% trimethylchlorosilane to the glass vial. Samples were incubated for 1 h at 60 °C and the mixture was transferred to a GC vial for analysis. Pooled samples were prepared by mixing 30 µL of derivatized metabolite extract from each sample to monitor the instrumental variations during the course of GC×GC-MS analysis. |
Combined analysis:
| Analysis ID | AN003348 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 6890N |
| Column | Agilent DB5-MS (60m × 0.25mm, 0.25um) |
| MS Type | EI |
| MS instrument type | GC x GC-TOF |
| MS instrument name | Leco Pegasus III GC TOF |
| Ion Mode | UNSPECIFIED |
| Units | intensity |
Chromatography:
| Chromatography ID: | CH002479 |
| Chromatography Summary: | The extracted and derivatized samples were analyzed on a LECO Pegasus GC×GC-TOF MS instrument (LECO Corp., St. Joseph, MI, USA) coupled to an Agilent 6890 gas chromatography and a Gerstel MPS2 autosampler (GERSTEL Inc., Linthicum, MD, USA), featuring a LECO two-stage cryogenic modulator and secondary oven. The primary column was a 60 m × 0.25 mm 1dc × 0.25 µm 1df DB-5 ms GC capillary column (phenyl arylene polymer virtually equivalent to (5%-phenyl)-methylpolysiloxane). The secondary GC column 1 m × 0.25 mm 2dc × 0.25 µm 2df, DB-17 ms ((50% phenyl)-methylpolysiloxane) was placed inside the secondary GC oven following the thermal modulator. Both columns were obtained from Agilent Technologies (Agilent Technologies J&W, Santa Clara, CA, USA) and were connected through a press fit connector. The helium carrier gas (99.999% purity) flow rate was set to 2.0 mL/min at a corrected constant flow via pressure ramps. The inlet temperature was set at 280 °C. The primary column temperature was programmed with an initial temperature of 60 °C for 0.5 min, then ramped at 5°C/min to 270 °C, and maintained for 15 min. The secondary column temperature program was set to an initial temperature of 70 °C for 0.5 min and then ramped at the same temperature gradient employed in the first column to 280 °C, accordingly. The thermal modulator was set to 15 °C relative to the primary oven and a modulation time was 2s. The mass range was set as 29-800 m/z with an acquisition rate of 200 mass spectra per second. The ion source chamber was 230°C with the transfer line temperature of 280°C, and the detector voltage was 1440 V with electron energy of 70 eV. The acceleration voltage was turned on after a solvent delay of 544 s, the split ratio was set at 10:1. |
| Instrument Name: | Agilent 6890N |
| Column Name: | Agilent DB5-MS (60m × 0.25mm, 0.25um) |
| Chromatography Type: | GC |
MS:
| MS ID: | MS003117 |
| Analysis ID: | AN003348 |
| Instrument Name: | Leco Pegasus III GC TOF |
| Instrument Type: | GC x GC-TOF |
| MS Type: | EI |
| MS Comments: | None |
| Ion Mode: | UNSPECIFIED |
