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MB Sample ID: SA217395
Local Sample ID: | Klb_KO_IF_5 |
Subject ID: | SU002349 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002349 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Klb_KO_IF_5 | SA217395 | FL026372 | Klb Knockout | Genotype |
Klb_KO_IF_5 | SA217395 | FL026372 | intermittent fasting | Treatment |
Collection:
Collection ID: | CO002342 |
Collection Summary: | Liver samples were collected from mice 3 weeks after intermittent fasting or ad libitum feeding. Samples included Control, Klb KO and Tbx3 KO livers. |
Sample Type: | Liver |
Treatment:
Treatment ID: | TR002361 |
Treatment Summary: | Liver samples were collected from mice 3 weeks after intermittent fasting or ad libitum feeding. Samples included Control, Klb KO and Tbx3 KO livers. |
Sample Preparation:
Sampleprep ID: | SP002355 |
Sampleprep Summary: | Liver specimens were harvested and immediately flash frozen in LN2 then stored at -80°C. While kept on dry ice a 20 mg sample was removed from each liver specimen, massed using an analytical balance, and placed in a 2 mL round bottom polypropylene tube containing 4-6, 2.3 mm stainless steel beads. 500 µL of -20°C extraction solution (methanol: acetonitrile: water, 2:2:1) containing stable isotope labeled metabolite standards was added to each sample tube. Ratio of 20 mg to 500 µL was retained when masses were not exactly 20 mg. All samples were homogenized at an amplitude of 20 Hz for 15 minutes and stored at -20°C for one hour to maximize protein precipitation. Samples were then vortexed for 20 seconds and centrifuged at 4°C for 5 minutes, speed 14,000 rcf. 120 µL of supernatant was removed from each tube and filtered using 0.2 µm polyvinylidene fluoride filter (Agilent Technologies P/N: 203980-100) and collected via 6,000 rcf centrifuge for 4 minutes. An additional 50uL was removed from each sample and combined into 5 pooled samples analyzed at equal intervals throughout the analysis to ensure stable signal. Extracts, pools, and procedural blanks were sealed and stored at 4°C until prompt analysis. |
Combined analysis:
Analysis ID | AN003696 | AN003697 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Thermo Vanquish | Thermo Vanquish |
Column | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | relative counts | relative counts |
Chromatography:
Chromatography ID: | CH002739 |
Chromatography Summary: | Untargeted metabolomics analysis was conducted as described previously (https://www.nature.com/articles/s41586-021-03707-9) with some modification. Liver extracts were analyzed via hydrophilic interaction liquid chromatography (HILIC) coupled to a Thermo Q-Exactive HF high resolution mass spectrometer. Each sample was analyzed in both positive and negative ionization modes (ESI+, ESI-) via subsequent injections. Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. Metabolomics data was processed using MS-DIAL v4.60 (https://www.nature.com/articles/s41587-020-0531-2) and queried against a combination of our in-house MS2 library (https://www.nature.com/articles/s41586-021-03707-9) and MassBank of North America, the largest freely available spectral repository (https://doi.org/10.1002/mas.21535). Annotations were scored using guidelines from the metabolomics standards initiative (https://www.nature.com/articles/nbt0807-846b). Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average. Statistical analysis of annotated features was implemented using MetaboAnalyst 5.0 (https://doi.org/10.1093/nar/gkab382). Data visualization including principal component analysis and volcano plots were generated using log10 transformed peak heights. |
Instrument Name: | Thermo Vanquish |
Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
Chromatography Type: | HILIC |
MS:
MS ID: | MS003447 |
Analysis ID: | AN003696 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. Metabolomics data was processed using MS-DIAL v4.60. Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average. |
Ion Mode: | POSITIVE |
Analysis Protocol File: | DeFelice_Methods.docx |
MS ID: | MS003448 |
Analysis ID: | AN003697 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. Metabolomics data was processed using MS-DIAL v4.60. Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average. |
Ion Mode: | NEGATIVE |
Analysis Protocol File: | DeFelice_Methods.docx |