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MB Sample ID: SA079505
| Local Sample ID: | Mouse 9 |
| Subject ID: | SU001208 |
| 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) |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001208 |
| 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 ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Mouse 9 | SA079505 | FL012190 | Antibiotics | Treament |
| Mouse 9 | SA079505 | FL012190 | Air | Exposure |
Collection:
| Collection ID: | CO001202 |
| 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. |
| Collection Protocol Filename: | PROTOCOL_AND_REPORT_150324.docx |
| Sample Type: | Lung |
Treatment:
| Treatment ID: | TR001223 |
| Treatment 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. |
| Treatment Protocol Filename: | PROTOCOL_AND_REPORT_150324.docx |
Sample Preparation:
| Sampleprep ID: | SP001216 |
| 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 | AN001882 | AN001883 | AN001884 | AN001885 |
|---|---|---|---|---|
| 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 UPLC BEH Amide (2.1x150 mm,1.7um) | Waters Acquity BEH C18 (100 x 2mm,1.7um)/Waters UPLC BEH Amide (2.1x150 mm,1.7um) | Waters Acquity BEH C18 (100 x 2mm,1.7um)/Waters UPLC BEH Amide (2.1x150 mm,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: | CH001363 |
| 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. |
| Methods Filename: | PROTOCOL_AND_REPORT_150324.docx |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH C18 (100 x 2mm,1.7um)/Waters UPLC BEH Amide (2.1x150 mm,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: | CH001364 |
| 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. |
| Methods Filename: | PROTOCOL_AND_REPORT_150324.docx |
| Instrument Name: | Thermo Trace DSQ |
| Column Name: | 5% diphenyl/95% dimethyl polysiloxane GC column |
| Chromatography Type: | GC |
MS:
| MS ID: | MS001738 |
| Analysis ID: | AN001882 |
| 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: | MS001739 |
| Analysis ID: | AN001883 |
| 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: | MS001740 |
| Analysis ID: | AN001884 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | HILIC LC-MS Negative |
| Ion Mode: | NEGATIVE |
| MS ID: | MS001741 |
| Analysis ID: | AN001885 |
| Instrument Name: | Thermo Trace DSQ |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | GC-MS |
| Ion Mode: | POSITIVE |
