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MB Sample ID: SA175950
Local Sample ID: | 5 |
Subject ID: | SU001970 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Male |
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Subject:
Subject ID: | SU001970 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Male |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
5 | SA175950 | FL021820 | WT_sham | Genotype_irradiation |
Collection:
Collection ID: | CO001963 |
Collection Summary: | Serum was collected after irradiation |
Sample Type: | Blood (serum) |
Treatment:
Treatment ID: | TR001982 |
Treatment Summary: | WT C57Bl/6 mice (C57BL/6NCrl strain code #027) were obtained from Charles River Laboratories (Frederick, MD) and DKI mice were kindly provided by the Laboratory of Immune Cell Biology, National Cancer Institute (P.I. Jonathan D. Ashwell, M.D.) (Jirmanova et al. 2011). Animals were bred/irradiated (12 h light / 12 h dark cycle conditions) at Georgetown University and water and food (PicoLab Rodent Diet 20 #5053) were provided ad libitum according to Georgetown University Institutional Animal Care and Use Committee (GUACUC) protocols (2016-1152). Before irradiation and biofluid collection the mice were acclimated to metabolic cages for 24 h. Male mice that were 8 – 10 weeks old were exposed to a total body ionization (TBI) x-ray dose (~1.67 Gy/min; X-Rad 320, Precision X-Ray Inc, Branford, CT; filter, 0.75 mm tin/ 0.25 mm copper/1.5 mm aluminum) of 0, 2, or 7 Gy. All urine samples were collected over a 24 h period in a metabolic cage pre-irradiation and at days 1, 3, and 7 d post-irradiation (Figure S1). Blood for metabolomics was collected at 1 d via cheek bleed from the submandibular vein and serum was separated in a BD microtainer serum separator tube and centrifuged for 10 min (10,000 x g, 4°C). Serum samples from sham-irradiated mice were used as a control (Figure S1). All biofluids were flash frozen and stored at -80°C until further use. Seven days post-irradiation, blood was collected in a dipotassium EDTA Tube (BD Cat #365974) via the facial vein from each animal and subjected to a complete blood count by VRL Diagnostics (Gaithersburg, MD, http://www.vrlsat.com/) (Figure S2). |
Sample Preparation:
Sampleprep ID: | SP001976 |
Sampleprep Summary: | Biofluids were prepared as previously described (Pannkuk et al. 2018;2020). Urine (20 μl) was deproteinated with 50% acetonitrile (80 μl) containing internal standards (2 μM debrisoquine sulfate, 30 μM 4-nitrobenzoic acid), incubated on ice for 10 min, vortexed for 30 seconds, and centrifuged for 10 min (10,000 x g, 4°C). Serum (5 μl) was prepared as above but was deproteinated with 66% acetonitrile (195 μl). A quality control (QC) sample was prepared by mixing 1 μl from each sample and prepared as above. |
Processing Storage Conditions: | -80℃ |
Combined analysis:
Analysis ID | AN003072 | AN003073 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Waters Acquity | Waters Acquity |
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 | QTOF | QTOF |
MS instrument name | Waters Synapt G2 | Waters Synapt G2 |
Ion Mode | POSITIVE | NEGATIVE |
Units | peak area | peak area |
Chromatography:
Chromatography ID: | CH002273 |
Chromatography Summary: | Mobile phases consisted of the following: solvent A (water/0.1% formic acid [FA]), solvent B (ACN/0.1% FA), solvent C (isopropanol [IPA]/ACN (90:10)/0.1% FA). The gradient for urine was (solvent A and B) 4.0 min 5% B, 4.0 min 20% B, 5.1 min 95% B, and 1.9 min 5% B at a flow rate of 0.5 ml/min. The gradient for serum was (solvent A, B, and C) 4.0 min 98:2 A:B, 4.0 min 40:60 A:B, 1.5 min 2:98 A:B, 2.0 min 2:98 A:C, 0.5 min 50:50 A:C, and 1.0 min 98:2 A:B at a flow rate of 0.5 ml/min. |
Instrument Name: | Waters Acquity |
Column Name: | Waters Acquity BEH C18 (50 x 2.1mm,1.7um) |
Flow Gradient: | The gradient for urine was (solvent A and B) 4.0 min 5% B, 4.0 min 20% B, 5.1 min 95% B, and 1.9 min 5% B at a flow rate of 0.5 ml/min. The gradient for serum was (solvent A, B, and C) 4.0 min 98:2 A:B, 4.0 min 40:60 A:B, 1.5 min 2:98 A:B, 2.0 min 2:98 A:C, 0.5 min 50:50 A:C, and 1.0 min 98:2 A:B at a flow rate of 0.5 ml/min. |
Flow Rate: | 0.5 ml/min |
Solvent A: | 100% water; 0.1% formic acid |
Solvent B: | solvent B:100% acetonitrile; 0.1% formic acid solvent C:90% isopropanol/10% acetonitrile; 0.1% formic acid |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS002859 |
Analysis ID: | AN003072 |
Instrument Name: | Waters Synapt G2 |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | Negative and positive electrospray ionization (ESI) data-independent modes were used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 1000 L/Hr. |
Ion Mode: | POSITIVE |
MS ID: | MS002860 |
Analysis ID: | AN003073 |
Instrument Name: | Waters Synapt G2 |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | Negative and positive electrospray ionization (ESI) data-independent modes were used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 1000 L/Hr. |
Ion Mode: | NEGATIVE |