Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA175979

Local Sample ID:	21
Subject ID:	SU001970
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

Select appropriate tab below to view additional metadata details:

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
21	SA175979	FL021823	dki_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).

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