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MB Sample ID: SA063454
| Local Sample ID: | M1 |
| Subject ID: | SU001047 |
| Subject Type: | Other |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001047 |
| Subject Type: | Other |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| M1 | SA063454 | FL010860 | Medium Dose Tempol | Treatment |
Collection:
| Collection ID: | CO001041 |
| Collection Summary: | 6-week-old wild-type male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) were transferred into anaerobic chamber (Coy Laboratory Products, Inc., Grass Lake, MI) following CO2 asphyxiation. All the following procedures were performed under strict anaerobic conditions with an oxygen level below 20 ppm. Cecal contents were collected |
| Sample Type: | Cecum |
Treatment:
| Treatment ID: | TR001061 |
| Treatment Summary: | The cecal content suspension was treated with tempol at a final concentration 0.01 mg/mL, 0.1 mg/mL and 1 mg/mL, following a brief centrifugation and incubation at 37 °C for 4 h in dark. |
Sample Preparation:
| Sampleprep ID: | SP001054 |
| Sampleprep Summary: | 600 µL of bacteria suspension after 4h incubation was centrifuged (700 g, 4 °C for 1 min) and supernatants were transferred to a new tube, centrifuged (6000 g, 4 °C for 3 min) and washed 3 times with PBS. After the final centrifugation, 1 mL cold 50% aqueous methanol containing 1 µM chlorpropamide and 1.0 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the microbial pellet, followed with homogenization (6500 rpm, 1 cycle, 60 s). The sample was freeze-thawed three times with liquid nitrogen to break apart tough microbial cell wall. Then the sample was centrifuged (max speed, 4 °C, and 10 min), supernatants were transferred to a new EP tube, dried down and resuspended in 200 µL 3% aqueous methanol. After a final spin (max speed, 4 °C, and 10 min), 150 µL supernatants were transferred to autosampler for LC-MS analysis. |
| Sampleprep Protocol ID: | LC sample prep.docx |
| Sampleprep Protocol Filename: | LC_sample_prep.docx |
Combined analysis:
| Analysis ID | AN001650 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Fisher Orbitrap Exactive plus |
| Column | Phenomenex (Torrance,CA) Hydro-RP C18 |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | ppm |
Chromatography:
| Chromatography ID: | CH001161 |
| Chromatography Summary: | Metabolomics profiling was performed with a Dionex Ultimate 3000 quaternary high-performance liquid chromatography (HPLC) pump, column compartment and autosampler coupled Exactive plus Orbitrap mass spectrometer controlled by Xcalibur 2.2 software (Thermo Fisher Scientific, Waltham, MA). LC-MS was run with a modified ion pairing reversed phase negative ion electrospray ionization method (61). A total volume of 10 µL sample is injected and separated on a Phenomenex (Torrance, CA) Hydro-RP C18 column (100 × 2.1 mm, 2.5 µm particle size) using a water/methanol gradient with tributylamine and acetic acid added to the aqueous mobile phase to enhance separation. The HPLC column is maintained at flow rate of 200 µL/min with the temperature of 30 °C. Solvents and gradient are as follows: Solvent A is 3% aqueous methanol with 10 mM tributylamine and 15 mM acetic acid; solvent B is 100% methanol. The gradient is 0 min, 0% B; 5 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5 min, 95% B; 19 min, 0% B; and 25 min, 0% B. The Exactive plus is operated in negative ion mode at maximum resolving power (140,000), and scanned from m/z 72 -1000 for the first 90 sec and then from m/z 85-1000 for the remainder of the chromatographic run for the detection of small molecule metabolites. The automatic gain control target is 3E6 with a maximum injection time of 100 us, the nitrogen sheath gas flow rate is set at 35, the auxillary gas at 10 and the sweep gas at 1. The capillary voltage is 3.2 kV and both the capillary and heater set at 200 °C, the S-lens was 55. |
| Instrument Name: | Thermo Fisher Orbitrap Exactive plus |
| Column Name: | Phenomenex (Torrance,CA) Hydro-RP C18 |
| Column Temperature: | 30 |
| Flow Gradient: | 0 min, 0% B; 5 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5 min, 95% B; 19 min, 0% B; and 25 min, 0% B. |
| Flow Rate: | 200 µL/min |
| Solvent A: | 97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001525 |
| Analysis ID: | AN001650 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
