Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA217403

Local Sample ID:	Tbx3_KO_IF_1
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
Tbx3_KO_IF_1	SA217403	FL026374	Tbx3 Knockout	Genotype
Tbx3_KO_IF_1	SA217403	FL026374	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.

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