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MB Sample ID: SA210066
| Local Sample ID: | LFP_363_S08 |
| Subject ID: | SU002272 |
| Subject Type: | Other organism |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6N mice |
| Age Or Age Range: | 5 weeks |
| Gender: | Male |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002272 |
| Subject Type: | Other organism |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6N mice |
| Age Or Age Range: | 5 weeks |
| Gender: | Male |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| LFP_363_S08 | SA210066 | FL025633 | LF diet + low dose penicillin (LFP) | Treatment |
Collection:
| Collection ID: | CO002265 |
| Collection Summary: | At the end of the experiment (5-weeks)after starting diet and antibiotic treatments, mice were humanely euthanized by CO2 administration. Afterwards, ileum (distal small intestine) content was collected. |
| Sample Type: | Feces |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002284 |
| Treatment Summary: | Animals were fed either a 60% fat diet (HF) (OpenSource Diets, #D12492) or a 10% fat control diet (LF) (OpenSource Diets, #D12450J) for 5 weeks. Groups of LF or HF diet mice were also given low doses of penicillin (Sigma Aldrich #P1382)(LDP) (6.67 mg/L, (10)) in their drinking water throughout the experiment or clinical doses of penicillin (0.167 g/L, (35)) in their drinking water from days 0 – 5, 15 – 20, and 30 – 35. For long-term experiments, mice were fed a HF diet or given a HF diet and LDP for 5 weeks before being switched to either a LF or HF diet alone for an additional 5 weeks. |
Sample Preparation:
| Sampleprep ID: | SP002278 |
| Sampleprep Summary: | Frozen mouse intestinal content samples (n=20, 5 biological replicates for each sample group) were lysed in 500 µl ice-cold lysis buffer (1:1:2, v:v:v, acetonitrile: methanol: ammonium bicarbonate 0.1M - pH 8.0) and sonicated individually using a probe tip sonicator at 50% power (10 pulses). The lysis buffer contained isotopically labeled standards (n=2) to determine sample process variability. Homogenized samples were normalized by weight to the smallest amount of tissue sample such that each sample contained an equal amount of tissue. Proteins were precipitated from individual samples by addition of 800 µL of ice-cold methanol followed by overnight incubation at -80°C. Precipitated proteins were pelleted by centrifugation (15,000 rpm, 15 min) and metabolite extracts were dried down in vacuo and stored at -80°C. Individual samples were reconstituted in 100 μL of reverse phase liquid chromatography reconstitution buffer (acetonitrile/water with 0.1% formic acid, 3:97, v/v) containing isotopically labeled standards (n=2) to assess instrument variability. A pooled quality control (QC) sample was prepared by pooling equal volumes (10 μL) from each individual sample following reconstitution. |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Following lysis and standard addition, protein precipitation was performed by adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 min to eliminate proteins. The supernatants containing metabolites were dried via speed-vacuum. |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003580 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Thermo Hypersil Gold (100 x 2. mm,1.9um) |
| MS Type | ESI |
| MS instrument type | QTRAP |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE |
| Units | time_m/z |
Chromatography:
| Chromatography ID: | CH002647 |
| Chromatography Summary: | Prepared samples were analyzed by RPLC-HRMS/MS in the Vanderbilt Center for Innovative Technology (CIT) using a modified version of a reversed phase chromatography negative ionization method. Metabolites were separated on a Thermo Fisher Scientific (Waltham, MA) Hypersil Gold C18 column (100 x 2.1 mm, 1.9 μm particle size) using water/acetonitrile gradient with formic acid (0.1%) added to both mobile phases. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Hypersil Gold (100 x 2. mm,1.9um) |
| Column Temperature: | 40 |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 80% acetonitrile/20% water; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003337 |
| Analysis ID: | AN003580 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | QTRAP |
| MS Type: | ESI |
| MS Comments: | Progenesis QI software |
| Ion Mode: | NEGATIVE |
