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MB Sample ID: SA071781
| Local Sample ID: | 1481_RV |
| Subject ID: | SU001103 |
| Subject Type: | Mammal |
| Subject Species: | Ovis aries |
| Taxonomy ID: | 9940 |
| Age Or Age Range: | 142 days gestation (fetal); Within 24 hours of birth (newborn) |
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Subject:
| Subject ID: | SU001103 |
| Subject Type: | Mammal |
| Subject Species: | Ovis aries |
| Taxonomy ID: | 9940 |
| Age Or Age Range: | 142 days gestation (fetal); Within 24 hours of birth (newborn) |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 1481_RV | SA071781 | FL011253 | Fetal | Age |
| 1481_RV | SA071781 | FL011253 | Female | Sex |
Collection:
| Collection ID: | CO001097 |
| Collection Summary: | Ewes were assigned to one of two groups: (1) a fetal group (n=7, 3 singleton and 2 multiple gestation pregnancies); (2) a newborn group (n=7, 2 singleton and 2 multiple gestation pregnancies). Heart tissue was collected from the right ventricle (RV), left ventricle (LV), and intraventricular septum (IVS) on the day of birth (newborn group) or at gestational day 142 (fetal group) and immediately frozen in liquid nitrogen. Samples were stored at -80 °C until data collection. |
| Sample Type: | Cardiac tissue |
| Collection Method: | Heart tissue was collected under sterile conditions and immediately frozen in liquid nitrogen |
| Collection Location: | University of Florida |
| Storage Conditions: | -80℃ |
| Collection Vials: | Cryovials |
| Storage Vials: | Cryovials |
Treatment:
| Treatment ID: | TR001117 |
| Treatment Summary: | NA |
Sample Preparation:
| Sampleprep ID: | SP001110 |
| Sampleprep Summary: | Homogenized right ventricle tissue samples (100 mg from 7 fetuses and 7 newborns) were used for lipid extraction via the Folch method. Briefly, 2 mL of internal standard mixture was added to each sample before adding 1.2 mL of 2:1 chloroform/methanol solution (HPLC-grade). Samples were incubated at 4 °C for 20 min with occasional vortexing. Following incubation, 200 mL of water (HPLC-grade) was added and samples were incubated at 4 °C for 10 min. Samples were centrifuged at 2,000 g for 5 min at 4 °C, and the resulting chloroform layer was removed and transferred into a clean tube. The extraction process was repeated with 400 mL 2:1 chloroform/methanol (HPLC-grade) and the resulting chloroform layers were combined. Samples were dried under a nitrogen stream at 30 °C and stored at -80 °C until reconstitution. Samples were reconstituted with 200 mL of isopropanol and 2 mL injection standard mixture. Ammonium acetate and all analytical grade solvents (formic acid, chloroform, and methanol) were purchased from Fisher Scientific (Waltham, MA, USA). All mobile phase solvents were Fisher Optima LC/MS-grade (acetonitrile, isopropanol, and water). |
| Processing Method: | 50 mg of tissue was weighed and added to tubes with three 3mm glass beads, two 5mm steal beads, and 0.7mm zirconia beads (“2 squirts”). HPLC-water (1mL) was added to each tube before homogenization at 1800 rpm for 30 seconds for 5 rounds. Samples were incubated at 4C for 20 min between rounds. Following homogenization, samples were centrifuged at 2,000g for 10 min at 4C to pellet tissue debris before transferring supernatant to a clean tube. Homogenates were stored at -80C. |
| Extraction Method: | 2 uL of internal standard mixture was added to each sample before adding 1.2 mL of 2:1 chloroform/methanol solution (HPLC-grade). Samples were incubated at 4 °C for 20 min with occasional vortexing. Following incubation, 200 mL of water (HPLC-grade) was added and samples were incubated at 4 °C for 10 min. Samples were centrifuged at 2,000 g for 5 min at 4 °C, and the resulting chloroform layer was removed and transferred into a clean tube. The extraction process was repeated with 400 mL 2:1 chloroform/methanol (HPLC-grade) and the resulting chloroform layers were combined. Samples were dried under a nitrogen stream at 30 °C (Organomation Associated MultiVap) and stored at -80 °C until reconstitution. |
| Sample Resuspension: | Samples were reconstituted with 200 mL of isopropanol and 2 mL injection standard mixture and vortexed to mix. Fifty uL of reconstituted sample was transferred to a glass LC vial for analysis. |
| Sample Spiking: | Internal standards: lysophosphatidylcholine (17:0), phosphatidylcholine (17:0/17:0), phosphatidylglycerol (14:0/14:0), phosphatidylethanolamine (15:0/15:0), phosphatidylserine (14:0/14:0), triglyceride (15:0/15:0/15:0). Injection Standards: lysophosphatidylcholine (19:0), phosphatidylcholine (19:0/19:0), phosphatidylglycerol (17:0/17:0), phosphatidylethanolamine (17:0/17:0), phosphatidylserine (17:0/17:0), triglyceride (17:0/17:0/17:0). |
Combined analysis:
| Analysis ID | AN001731 | AN001732 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | Waters Acquity BEH C18 (50 x 2.1mm,1.7um) | Waters Acquity BEH C18 (50 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Relative peak area | Relative peak area |
Chromatography:
| Chromatography ID: | CH001224 |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters Acquity BEH C18 (50 x 2.1mm,1.7um) |
| Column Temperature: | 4C |
| Flow Gradient: | 80% A and 20% B at 0 min; 80% A and 20% B at 1 min; 70% A and 30% B at 3 min; 55% A and 45% B at 4 min; 40% A and 60% B at 6 min; 35% A and 65% B at 8 min; 35% A and 65% B at 10 min; 10% A and 90% B at 15 min; 2% A and 98% B at 17 min; 2% A and 98% B at 18 min; 80% A and 20% B at 19 min; 80% A and 20% B at 23 min |
| Flow Rate: | 0.5mL/min |
| Sample Injection: | 2 uL |
| Solvent A: | 40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/8% acetonitrile/2% water; 0.1% formic acid; 10 mM ammonium formate |
| Analytical Time: | 18 min |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001600 |
| Analysis ID: | AN001731 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 250C |
| Ionization: | Heated electrospray |
| Spray Voltage: | 1500 |
| Resolution Setting: | 70,000 |
| Scan Range Moverz: | 200-2200 m/z |
| MS ID: | MS001601 |
| Analysis ID: | AN001732 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 250C |
| Ionization: | Heated electrospray |
| Spray Voltage: | 1500 |
| Resolution Setting: | 70,000 |
| Scan Range Moverz: | 200-2200 m/z |
