Summary of Study ST001712

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001096. The data can be accessed directly via it's Project DOI: 10.21228/M81M6P This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study IDST001712
Study TitleMetabolomics analysis of plasma from a mouse model of astrocytoma subjected to radiotherapy
Study SummaryMice were randomized in two groups (n=9 mice/group), one group was subjected to radiotherapy (Monday and Friday for 2 consecutive weeks at 3Gy/session) and the other cohort was the control. Sample were taken approximately each 10 days from the tail vein.
Institute
National Cancer Institute
Last NameLarion
First NameMioara
Address37 Convent Dr, Building 37 Room 1136
Emailmioara.larion@nih.gov
Phone2407606825
Submit Date2021-02-26
Analysis Type DetailLC-MS
Release Date2021-03-09
Release Version1
Mioara Larion Mioara Larion
https://dx.doi.org/10.21228/M81M6P
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001096
Project DOI:doi: 10.21228/M81M6P
Project Title:Plasma biomarkers of radiotherapy in gliomas
Project Summary:Metabolomics analysis of plasma collected from a mouse model of astrocytoma
Institute:NIH
Department:Neuro-Oncology
Laboratory:Cancer Metabolism
Last Name:Ruiz Rodado
First Name:Victor
Address:National Cancer Institute Building 37,, Room 1136, Bethesda, MD, 20892, USA
Email:victor.ruizrodado@nih.gov
Phone:2028736662

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
Species Group:Mammals

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

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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.

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.

Combined analysis:

Analysis ID AN002787
Chromatography ID CH002062
MS ID MS002583
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
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