Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA160370

Local Sample ID:	2205
Subject ID:	SU001789
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	6–8 weeks old
Gender:	Female
Animal Animal Supplier:	Charles River Frederick Research Model Facility

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Subject:

Subject ID:	SU001789
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	6–8 weeks old
Gender:	Female
Animal Animal Supplier:	Charles River Frederick Research Model Facility

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
2205	SA160370	FL018803	RT	Treatment
2205	SA160370	FL018803	RT+4	Time
2205	SA160370	FL018803	RT	Treatment
2205	SA160370	FL018803	RT+4	Time

Collection:

Collection ID:	CO001782
Collection Summary:	Blood was collected approximately every 10 days from the tail vein of the mice in Li-heparin collection tubes and immediately processed to separate plasma.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR001802
Treatment Summary:	Radiation was performed on mice intracranially injected with the NCH1681 cell line. Mice were irradiated with a total of 12Gy; specifically, animals were treated on Monday and Friday for 2 consecutive weeks at 3Gy/session. Radiation was performed in a Pantek machine an orthovoltage radiotherapy unit. The mice were anesthetized with a cocktail of ketamine/rompun/saline mixture, i.e. ketamine (100mg/ml), rompun (20mg/ml) and diluted with saline to give the mice a 100mg/kg dose of ketamine and 10mg/kg rompun. The mice were injected with the cocktail at a dose of 0.01 µL per gram of half of the mouse’s body weight. Animals were then placed in a custom-made jig. During radiation, lead shielding covered the eyes, ears, the oral cavity, and the spinal cord. After radiation, the mice were given atipamezole, a reversal agent, to aid in the recovery.

Treatment ID:

TR001802

Treatment Summary:

Radiation was performed on mice intracranially injected with the NCH1681 cell line. Mice were irradiated with a total of 12Gy; specifically, animals were treated on Monday and Friday for 2 consecutive weeks at 3Gy/session. Radiation was performed in a Pantek machine an orthovoltage radiotherapy unit. The mice were anesthetized with a cocktail of ketamine/rompun/saline mixture, i.e. ketamine (100mg/ml), rompun (20mg/ml) and diluted with saline to give the mice a 100mg/kg dose of ketamine and 10mg/kg rompun. The mice were injected with the cocktail at a dose of 0.01 µL per gram of half of the mouse’s body weight. Animals were then placed in a custom-made jig. During radiation, lead shielding covered the eyes, ears, the oral cavity, and the spinal cord. After radiation, the mice were given atipamezole, a reversal agent, to aid in the recovery.

Sample Preparation:

Sampleprep ID:	SP001795
Sampleprep Summary:	Blood samples were separated into plasma and packed cells by centrifugation at 3,500g for 15 min at 4°C and stored at -80°C until extraction. 35 µL of plasma were extracted in a water:methanol:chloroform mixture. Centrifuged for 20 min at 4°ᵒC and 13,000 rpm and the resulting upper hydrophilic phase was then transferred to a clean vial and dried under a stream of N2 gas. Dried sediments were resuspended in 60% methanol (aq.) and injected into the LCMS system.

Sampleprep ID:

SP001795

Sampleprep Summary:

Blood samples were separated into plasma and packed cells by centrifugation at 3,500g for 15 min at 4°C and stored at -80°C until extraction. 35 µL of plasma were extracted in a water:methanol:chloroform mixture. Centrifuged for 20 min at 4°ᵒC and 13,000 rpm and the resulting upper hydrophilic phase was then transferred to a clean vial and dried under a stream of N2 gas. Dried sediments were resuspended in 60% methanol (aq.) and injected into the LCMS system.

Combined analysis:

Analysis ID	AN002787
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Agilent 6545 QTOF-MS
Column	AdvanceBio Glycan Map (2.1 x 100mm, 2.7µm)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Agilent 6540 QTOF
Ion Mode	NEGATIVE
Units	arbitrary units

Chromatography:

Chromatography ID:	CH002062
Instrument Name:	Agilent 6545 QTOF-MS
Column Name:	AdvanceBio Glycan Map (2.1 x 100mm, 2.7µm)
Column Temperature:	35
Flow Gradient:	100% B, 0.5 min; 95% B, 2.0 min; 60% B, 3.0 min; 35% B, 5 min; hold 0.25 min; 0% B, 6 min; hold 0.5 min; 100% B, 7.8 min; equilibrate for 1.7 min.
Flow Rate:	0.2 mL/min
Solvent A:	88% water/12% acetonitrile; 10 mM ammonium acetate; titrated with formic acid and ammonium hydroxide to pH 6.85
Solvent B:	90% acetonitrile/10% water; 10 mM ammonium acetate; titrated with formic acid and ammonium hydroxide to pH 6.85
Chromatography Type:	HILIC

MS:

MS ID:	MS002583
Analysis ID:	AN002787
Instrument Name:	Agilent 6540 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	LC/MS analysis was conducted with the Agilent 6545 QTOF-MS combined with 1290 Infinity II UHPLC system (Agilent Technologies, Wilmington, DE, USA). Only LC/MS grade solvents and additives purchased from Covachem (CovaChem, LLC., Loves Park, IL, USA) were used to prepare mobile phases and wash solutions. Wash cycles consisting of strong wash (50% Methanol, 25% Isopropanol, and 25% Water), weak wash (90% Acetonitrile and 10 % Water), and seal wash (10% Isopropanol and 90% water) were implemented to eliminate carryover between injections. Dried extracts were reconstituted in 80 µL 60:40 MeOH/H2O and samples were injected (8 µL) to resolve analytes using Infinity 1290 in-line filter combined with AdvanceBio Glycan Map 2.1 x 100mm, 2.7µm column (Agilent Technologies, Wilmington, DE., USA) set at 35 0C. The solvent buffers were composed of mobile phase A (10 mM ammonium acetate in 88% water/ 12% acetonitrile) and mobile phase B (10 mM ammonium acetate in 90 % Acetonitrile) titrated with formic acid and ammonium hydroxide to pH 6.85. The linear gradient was executed at flow rate 0.2 mL/min, as follows: 100 % B, 0.5 min; 95% B, 2.0 min; 60 % B, 3.0 min; 35 % B, 5 min; hold 0.25 min; 0% B, 6 min; hold 0.5 min; 100 % B, 7.8 min; equilibrate for 1.7 min. The mass analyzer acquisition parameters include drying gas temperature, 250 0C; drying gas flow, 9 L/min; sheath gas temperature, 325 0C; sheath gas flow, 11 L/min; nebulizer, 45 psig. Mass spectra were acquired at 3.0 spectra/s in negative electrospray ionization (ESI-) mode for a mass range from 72 to 1200 m/z using a voltage gradient of capillary 3000 V, nozzle 2000 V, fragmentor 80 V, skimmer 50 V, and octopole radio frequency 750 V.
Ion Mode:	NEGATIVE