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MB Sample ID: SA097894
| Local Sample ID: | AR424_Low_T_THR1plus4_8pt3 |
| Subject ID: | SU001422 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57Bl/6JMarp |
| Age Or Age Range: | 7 to 12 weeks |
| Gender: | Male |
| Animal Animal Supplier: | Monash University Animal Research Platform, Clayton, AUS |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001422 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57Bl/6JMarp |
| Age Or Age Range: | 7 to 12 weeks |
| Gender: | Male |
| Animal Animal Supplier: | Monash University Animal Research Platform, Clayton, AUS |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| AR424_Low_T_THR1plus4_8pt3 | SA097894 | FL013732 | Low Threonine diet SF18-066 | Diet |
| AR424_Low_T_THR1plus4_8pt3 | SA097894 | FL013732 | AAV-yTHR1+THR4 | Virus treatment |
Collection:
| Collection ID: | CO001417 |
| Collection Summary: | Mice were humanely euthanised for tissue collection |
| Sample Type: | Liver |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001437 |
| Treatment Summary: | Male mice aged 7 weeks upon arrival, were acclimated to the local housing facility (12-12h light-dark cycle, 22-24°C) for one week prior to experimentation and were fed standard rodent chow (8720610, Barastoc, AUS). Following acclimation, mice were administered a total of 5E+11 virus particles per mouse via the tail vein. For the negative control (NC): 5E+11 GFP-AAV; and for the THR1/4 overexpression studies mice were administered 2.5E+11 virus particles each of yTHR1-AAV and yTHR4-AAV. One week following this time, the dietary intervention was initiated. In these studies, the low threonine diet (SF18-066, Specialty Feeds AUS) contained homoserine, the substrate of THR1. The corresponding control diet was SF17-177 (Specialty Feeds AUS). |
Sample Preparation:
| Sampleprep ID: | SP001430 |
| Sampleprep Summary: | Livers were cryogenically pulverized (cryopulverization) using a 12-well biopulverizer (BioSpec Products, OK USA Part number 59012MS) according to the manufacturer’s instructions. The frozen tissue powder was then weighed and extracted in 20 µL of extraction solvent (0°C) per mg of tissue. The mixture was then briefly vortexed before sonication in an ice-water bath for 10 minutes followed by centrifugation (20,000 rcf, 4℃, 10 minutes). The supernatant was then transferred to a mass spectrometry vial for LC-MS analysis. The extraction solvent consisted of 2:6:1 CHCl3:MeOH:H2O v/v/v with 2 µM CHAPS, CAPS, PIPES and TRIS as internal standards. Additionally where quantitative amino acid analysis was performed, a mixture of 17 stable isotope labelled amino acids (Gln, Asn and Trp were absent) were added at a concentration of 500 pmol of each amino acid per mg liver (Cambridge Isotope Laboratories PN MSK-A2-1.2). |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN002242 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RS |
| Column | SeQuant ZIC-pHILIC (150 x 4.6mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Peak Area |
Chromatography:
| Chromatography ID: | CH001648 |
| Chromatography Summary: | Chromatography utilized a ZIC-pHILIC column (column temperature 25 °C) with a gradient elution of 20 mM ammonium carbonate (A) and acetonitrile (B) (linear gradient time-%B as follows: 0 min-80%, 15 min-50%, 18 min-5%, 21 min-5%, 24 min-80%, 32 min-80%) on a Dionex RSLC3000 UHPLC (Thermo). The flow rate was maintained at 300 μL/min. Samples were kept at 4 °C in the autosampler and 10 μL injected for analysis. |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | SeQuant ZIC-pHILIC (150 x 4.6mm,5um) |
| Column Temperature: | 25 |
| Flow Gradient: | linear gradient time-%B as follows: 0 min-80%, 15 min-50%, 18 min-5%, 21 min-5%, 24 min-80%, 32 min-80% |
| Flow Rate: | 300 µL/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% water; acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002088 |
| Analysis ID: | AN002242 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry was performed at 35 000 resolution (accuracy calibrated to <1 ppm) on a Q-Exactive Orbitrap MS (Thermo) operating in rapid switching positive (4 kV) and negative (−3.5 kV) mode electrospray ionization (capillary temperature 300 °C; sheath gas 50; Aux gas 20; sweep gas 2; probe temp 120 °C). |
| Ion Mode: | UNSPECIFIED |
