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MB Sample ID: SA079546

Local Sample ID:Mouse 28
Subject ID:SU001209
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:8 weeks
Gender:Male
Animal Animal Supplier:Taconic Farms (New York)

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Subject:

Subject ID:SU001209
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:8 weeks
Gender:Male
Animal Animal Supplier:Taconic Farms (New York)

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Mouse 28SA079546FL012195AntibioticsTreament
Mouse 28SA079546FL012195OzoneExposure

Collection:

Collection ID:CO001203
Collection Summary:Global biochemical profiles were determined in lung tissue and serum collected from untreated control mice and mice treated for two weeks with untreated drinking water or water containing an antibiotic cocktail (ampicillin, neomycin, metronidazole, and vancomycin), followed by a three hour exposure to ambient air or ozone (2ppm). Sample collection occurred 24 hours post-ozone exposure. Both serum and lung tissue were collected and processed from the same mouse.
Sample Type:Blood (serum)

Treatment:

Treatment ID:TR001224
Treatment Summary:Global biochemical profiles were determined in serum collected from untreated control mice and mice treated for two weeks with untreated drinking water or water containing an antibiotic cocktail (ampicillin, neomycin, metronidazole, and vancomycin), followed by a three hour exposure to ambient air or ozone (2ppm). Sample collection occurred 24 hours post-ozone exposure.

Sample Preparation:

Sampleprep ID:SP001217
Sampleprep Summary:Sample preparation: Samples were shipped to Metabolon for processing and prepared for metabolomics as previously described. Briefly, an equivalent amount serum (100µl) on a per sample basis was prepared using the automated MicroLab STAR® system from Hamilton Company. For QC purposes, a recovery standard was added prior to the first step in the extraction process. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were first precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions as follows. One fraction was used for analysis by UPLC-MS/MS with positive ion mode electrospray ionization. Another fraction was used for analysis by UPLC-MS/MS with negative ion mode electrospray ionization. The third and fourth fractions were used for LC polar platform, and for analysis by GC-MS. The final fraction was reserved as a backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. For LC, the samples were stored overnight under nitrogen before preparation for analysis. For GC, each sample was dried under vacuum overnight before preparation for analysis.

Combined analysis:

Analysis ID AN001886 AN001887 AN001888 AN001889
Analysis type MS MS MS MS
Chromatography type Reversed phase/HILIC Reversed phase/HILIC Reversed phase/HILIC GC
Chromatography system Waters Acquity Waters Acquity Waters Acquity Thermo Trace DSQ
Column Waters Acquity BEH C18 (100 x 2mm,1.7um) Waters Acquity BEH C18 (100 x 2mm,1.7um) Waters Acquity BEH C18 (100 x 2mm,1.7um) 5% diphenyl/95% dimethyl polysiloxane GC column
MS Type ESI ESI ESI EI
MS instrument type Orbitrap Orbitrap Orbitrap Single quadrupole
MS instrument name Thermo Q Exactive Plus Orbitrap Thermo Q Exactive Plus Orbitrap Thermo Q Exactive Plus Orbitrap Thermo Trace DSQ
Ion Mode POSITIVE NEGATIVE NEGATIVE POSITIVE
Units area under the curve area under the curve area under the curve area under the curve

Chromatography:

Chromatography ID:CH001365
Chromatography Summary:The LC/MS portion of the platform was based on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. Each sample extract was first dried, then reconstituted in acidic or basic LC-compatible solvents. To ensure injection and chromatographic consistency, these solvents each contained 8 or more injection standards at fixed concentrations. One aliquot was analyzed using acidic positive ion optimized conditions and the other using basic negative ion optimized conditions in two independent injections using separate dedicated columns (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm). Extracts reconstituted in acidic conditions were gradient eluted from a C18 column using water and methanol containing 0.1% formic acid. The basic extracts were similarly eluted from C18 using methanol and water, however with 6.5mM Ammonium Bicarbonate. The third aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate. The MS analysis alternated between MS and data-dependent MS2 scans using dynamic exclusion, and the scan range was from 80-1000 m/z.
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2mm,1.7um)
Solvent A:100% water; 0.1% formic acid
Solvent B: 100% methanol; 0.1% formic acid
Chromatography Type:Reversed phase/HILIC
  
Chromatography ID:CH001366
Chromatography Summary:For GC-MS analysis, samples were dried under vacuum for at least 18 h prior to derivatization under dried nitrogen using bistrimethyl-silyltrifluoroacetamide. Derivatized samples were separated on a 5% diphenyl / 95% dimethyl polysiloxane fused silica column (20 m x 0.18 mm ID; 0.18 um film thickness) with helium as carrier gas and a temperature ramp from 60° to 340°C over a 17.5 min period. Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer using electron impact ionization (EI) and operated at unit mass resolving power. The scan range was from 50–750 m/z.
Instrument Name:Thermo Trace DSQ
Column Name:5% diphenyl/95% dimethyl polysiloxane GC column
Chromatography Type:GC

MS:

MS ID:MS001742
Analysis ID:AN001886
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Reversed phase LC-MS Positive
Ion Mode:POSITIVE
  
MS ID:MS001743
Analysis ID:AN001887
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Reversed phase LC-MS Negative
Ion Mode:NEGATIVE
  
MS ID:MS001744
Analysis ID:AN001888
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:HILIC LC-MS Negative
Ion Mode:NEGATIVE
  
MS ID:MS001745
Analysis ID:AN001889
Instrument Name:Thermo Trace DSQ
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:GC-MS
Ion Mode:POSITIVE
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