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MB Sample ID: SA315468
| Local Sample ID: | D3_4 |
| Subject ID: | SU003004 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Age Or Age Range: | 15-20 week old |
| Gender: | Male |
| Species Group: | C57BL/6J |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003004 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Age Or Age Range: | 15-20 week old |
| Gender: | Male |
| Species Group: | C57BL/6J |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| D3_4 | SA315468 | FL037517 | Day 3 | Gln Treatment |
Collection:
| Collection ID: | CO002997 |
| Collection Summary: | MI was induced in adult (15-20 week old) male C57BL/6J mice as previously described.2,4,6 Mice were anesthetized (2% isoflurane) and laid supine on a heated stage (37°C), intubated and connected to a mouse ventilator (Harvard Apparatus; 300µL stroke volume, 300 breaths/min). The chest was incised to expose the ribs, and the heart exposed between the 3rd and 4th ribs. The pericardial layer was gently removed, and the left coronary artery was ligated ~1mm below the left atrium using 8-0 suture. Ischemia was confirmed by blanching of the LV. For glutamine administration, mice were injected intraperitoneally with glutamine (0.75-1g/kg) or saline vehicle beginning at either day 1 post-MI (for day 3 time-point) or beginning at day 3 (for day 7 time-point). A separate cohort of mice was injected 2 hr after MI and studied 24 hr later (day 1). To inhibit glutamine metabolism, mice were administered BPTES (12.5mg/kg; 10% in DMSO/corn oil), a glutaminase-1 inhibitor, beginning at either day 1 or day 3. |
| Sample Type: | Macrophages |
Treatment:
| Treatment ID: | TR003013 |
| Treatment Summary: | For glutamine administration, mice were injected intraperitoneally with glutamine (0.75-1g/kg) or saline vehicle beginning at either day 1 post-MI (for day 3 time-point) or beginning at day 3 (for day 7 time-point). A separate cohort of mice was injected 2 hr after MI and studied 24 hr later (day 1). To inhibit glutamine metabolism, mice were administered BPTES (12.5mg/kg; 10% in DMSO/corn oil), a glutaminase-1 inhibitor, beginning at either day 1 or day 3. |
Sample Preparation:
| Sampleprep ID: | SP003010 |
| Sampleprep Summary: | Frozen samples were stored at -80°C until analyzed by LC-MS-based metabolomics in the Vanderbilt Center for Innovative Technology (CIT). Isotopically labeled phenylalanine-D8 and biotin-D2 were added to 200 μL of culture supernatant per sample, and protein was precipitated by addition of 800 μL of ice-cold methanol followed by overnight incubation at −80°C. Precipitated proteins were pelleted by centrifugation (15,000 rpm, 15 min), and extracted metabolites were dried down in vacuo and stored at −80°C. Individual samples were reconstituted in 100 μL of reconstitution buffer (acetonitrile/water, 90:10, vol/vol) containing tryptophan-D3 and inosine-4N15. Equal volumes of individual samples were pooled to create a quality control (QC) pooled sample used for column conditioning, retention time alignment, to assess instrument reproducibility, and for batch acceptance. |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN004750 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Vanquish UHPLC |
| Column | ACQUITY UPLC BEH Amide HILIC |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE |
| Units | time_m/z |
Chromatography:
| Chromatography ID: | CH003582 |
| Chromatography Summary: | Metabolite extracts were separated on ACQUITY UPLC BEH Amide HILIC 1.7 μm, 2.1 × 100 mm column (Waters Corporation, Milford, MA) held at 30°C. Liquid chromatography was performed at a 200 μL min using solvent A (5 mM Ammonium formate in 90% water, 10% acetonitrile, and 0.1% formic acid) and solvent B (5 mM Ammonium formate in 90% acetonitrile, 10% water, and 0.1% formic acid) with a gradient length of 30 min. |
| Instrument Name: | Vanquish UHPLC |
| Column Name: | ACQUITY UPLC BEH Amide HILIC |
| Column Temperature: | 30 |
| Flow Gradient: | Linear gradient of 30 min |
| Flow Rate: | 0.20mL/min |
| Solvent A: | 90% water/10% acetonitrile; 5mM ammonium formate; 0.1% formic acid |
| Solvent B: | 10% water/90% acetonitrile; 5mM ammonium formate; 0.1% formic acid |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004496 |
| Analysis ID: | AN004750 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The acquired raw data were imported, processed, normalized and reviewed using Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK). All MS and MS/MS sample runs were aligned against a QC pool reference run, and unique ions (retention time and m/z pairs) were deadducted and deisotoped to generate unique "features" (retention time and m/z pairs). Data were normalized to all features using Progenesis QI. Experimental data annotations were assigned based on consistent retention time and MS2 fragmentation pattern matches with reference standards. |
| Ion Mode: | NEGATIVE |
