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MB Sample ID: SA308641
| Local Sample ID: | WT_Liver_MR08 |
| Subject ID: | SU002963 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002963 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| WT_Liver_MR08 | SA308641 | FL037164 | lung | Organism part |
Collection:
| Collection ID: | CO002956 |
| Collection Summary: | Briefly, 6-8-week-old female NOD/SCID/IL2gR mice (005557 Jackson Laboratory) were injected via tail vein with 1 x 10^5 A375 human melanoma cell line of indicated genotypes, n=5/group. Mice were monitored over 3-4 weeks. When mice showed signs of sickness, 20% of weight loss, impaired activity, hunching and limb paralysis, animals were euthanized, and organs were harvested for evaluation on metastasis presence, liver and lung metastasis were snap frozen and subjected to downstream analysis. |
| Sample Type: | Biopsy |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002972 |
| Treatment Summary: | Briefly, 6-8-week-old female NOD/SCID/IL2gR mice (005557 Jackson Laboratory) were injected via tail vein with 1 x 10^5 A375 human melanoma cell line of indicated genotypes, n=5/group. |
| Animal Endp Euthanasia: | When mice showed signs of sickness, 20% of weight loss, impaired activity, hunching and limb paralysis. |
Sample Preparation:
| Sampleprep ID: | SP002969 |
| Sampleprep Summary: | Metabolites were extracted by a two-step liquid extraction adapted from Sellik et al.[1], 50 mg of frozen tissue was used and 500 µL of prechilled methanol (-80 °C) was added. For quantification and data normalization, 25 µL of internal standard solution containing 13C6-L-Arginine, 13C5 L Valine, 13C2-Citric acid, 2H4-Succinic acid and 13C6-Fructose-6-phosphate at a concentration of 100 µM was added. This results in a concentration of 10 µM in the final extract. The sample was homogenized by a tissue slicer and the extract was vortexed for 2 min until no larger cell clusters were visible. Afterward, the sample was sonicated for 2 min in a chilled (0 °C) ultrasonic bath. The cells were kept at -80 °C for 5 min and subsequently thawed. The thawed cells were vortexed and sonicated for 2 min each and centrifuged at 3000 x g for 5 min. The supernatant was collected and 250 µL of water acidified with 0.1 % acetic acid (LC-MS grade) was added. The sample was vortexed and sonicated for 2 min and subject to the same freezing-thawing cycle described before. The samples were centrifuged at 3000 x g for 5 min and the supernatant was collected. The combined supernatant was dried in a vacuum-centrifuge for 45 min. The residue was resuspended in 250 µL acetonitrile/water (50/50; v/v) by sonicating and vortexing for 2 min each. To remove remaining proteins or other cell debris the extracts have been centrifuged at 12,000 rpm for 2 min and filtered using Nanosep 3KDa Omega centrifugal filters (Pall, Port Washington, USA). After the extraction pooled QC samples were generated for the whole sample set as well as for each investigated sample group and analyzed along the study samples to monitor the analytical quality. Ref: [1] (Sellick CA, Hansen R, Stephens GM, Goodacre R, Dickson AJ. Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling. Nat Protoc. 2011 28;6(8):1241-9. doi: 10.1038/nprot.2011.366) |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN004670 | AN004671 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Agilent 1290 Infinity II Bio LC system | Agilent 1290 Infinity II Bio LC system |
| Column | Agilent AdvanceBio MS Spent Media (150 x 2.1mm, 2.7um) | Agilent AdvanceBio MS Spent Media (150 x 2.1mm, 2.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak Area | Peak Area |
Chromatography:
| Chromatography ID: | CH003515 |
| Chromatography Summary: | For liquid chromatography, an Agilent 1290 Infinity II Bio LC system (Agilent Technologies Inc., Waldbronn, Germany) consisting of a 1290 Bio High-Speed Pump, a 1290 Bio Multisampler and a 1290 MCT with an AdvanceBio MS Spent Media (150 x 2.1 mm, 2.7 µm; Agilent Technologies Inc., Waldbronn, Germany) were used. The elution was carried out by utilizing a gradient at a flow rate of 250 μL/min with water (10 mM ammonium acetate pH 9) as solvent A and acetonitrile/water (90/10; v/v; 10 mM ammonium acetate pH 9) as solvent B. The linear gradient was: 0 min, 90% B; 2 min, 90% B; 12 min, 40% B; 13 min, 20% B; 16 min, 20% B; followed by 9 min at initial condition for re-equilibration. Column temperature was 35 °C, injection volume 5 µL. |
| Instrument Name: | Agilent 1290 Infinity II Bio LC system |
| Column Name: | Agilent AdvanceBio MS Spent Media (150 x 2.1mm, 2.7um) |
| Column Temperature: | 35 °C |
| Flow Gradient: | none |
| Flow Rate: | 250 μL/min |
| Solvent A: | water (10 mM ammonium acetate pH 9) |
| Solvent B: | acetonitrile/water (90/10; v/v; 10 mM ammonium acetate pH 9) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004417 |
| Analysis ID: | AN004670 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Data were analyszed in both positive and negative ion mode separately. MS-Dial version 4.9 was used for feature analysis. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004418 |
| Analysis ID: | AN004671 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Data were analyszed in both positive and negative ion mode separately. MS-Dial version 4.9 was used for feature analysis. |
| Ion Mode: | NEGATIVE |
