Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA332592

Local Sample ID:	Kidney494
Subject ID:	SU003192
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU003192
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Kidney494	SA332592	FL039358	water	Treatment

Collection:

Collection ID:	CO003185
Collection Summary:	Blood was collected into tubes containing EDTA anticoagulant via cardiac puncture. Plasma was obtained by centrifugation of whole blood at 1,500 g for 10 min at 4 °C and stored at -80 °C freezer.
Sample Type:	plasma, adipose, liver, muscle, heart, kidney, intestine, adipose

Treatment:

Treatment ID:	TR003201
Treatment Summary:	C57BL/6 mice were housed North Carolina State University Biological Resources Facility with ad libitum access to food (Laboratory Rodent Diet 5001) and water on a 12-hour light/dark cycle. At the age of 8 weeks, mice were administrated metformin in their drinking water (1mg/ml) for 12 days. Before tissue and blood collection, mice were fasted for 5 hours (6am-11am). Mice were then anesthetized with isoflurane and sacrificed via cervical dislocation.

Treatment ID:

TR003201

Treatment Summary:

C57BL/6 mice were housed North Carolina State University Biological Resources Facility with ad libitum access to food (Laboratory Rodent Diet 5001) and water on a 12-hour light/dark cycle. At the age of 8 weeks, mice were administrated metformin in their drinking water (1mg/ml) for 12 days. Before tissue and blood collection, mice were fasted for 5 hours (6am-11am). Mice were then anesthetized with isoflurane and sacrificed via cervical dislocation.

Sample Preparation:

Sampleprep ID:	SP003198
Sampleprep Summary:	All tissue sample was first homogenized in liquid nitrogen and then 10-20 mg tissues were weighed into a new 1.7 ml Eppendorf tube. Ice cold extraction solvent (400 ul 80% methanol/water) and 10 ul metformin-d6 (50 ng/ul in water) was added to each sample. Geno/Grinder homogenizer was used (1500 rpm, 1 to 2 min) to further break down the tissue chunk and form an even suspension. 200 ul supernatant containing polar metabolites was removed after centrifugation at 20,000 g at 4 °C for 10 min and transferred to LC vial for polar metabolite analysis using LC-HRMS. The rest samples (200 ul solvent and insoluble pellet) were briefly homogenized using Geno/Grinder again (1500 rpm, 30 sec) to loosen the bottom pellet and better mix with extraction solvent. 480 ul MTBE and 10 ul internal standard solution were added. After rigorous vortexing, 120 ul water was added to initiate phase separation. All samples were centrifuged at 20,000 g at 4 °C for 10 min. The supernatant containing lipids was transferred to a new Eppendorf tube and dried using speed vacuum. Dry pellets were stored in -80 °C freezer until ready for LC-HRMS analysis. To extract metabolites and lipids from mouse plasma, 10 ul plasma was mixed with 10 l water containing internal standards (50 ng/ul metformin-d6, 5 mM [U-13C]-glucose and [U-13C]-lactate, and 80 ul ice cold methanol was added. After vortex for 1 min, the mixture was centrifuged with a speed of 20,000 g at 4 °C for 10 min, and 20 ul was transferred directly to LC vial without solvent evaporation, followed by LC-HRMS analysis (injection volume, 3 ul) of polar metabolites such as glucose and metformin. To the rest samples (80 l solvent and pellet), 192 ul MTBE and 10 ul lipid internal standard solution were added. After rigorous vortexing, 48 ul water was added to initiate phase separation. All samples were centrifuged at 20,000 g at 4 °C for 10 min. The supernatant containing lipids was transferred to a new Eppendorf tube and dried using speed vacuum. The dry pellets of lipids from mouse tissues or plasma were reconstituted into (300 ul for tissue and 50 ul for plasma) sample solvent (isopropanol: ethyl acetate, 1:1, v/v), and 3 ul was injected to LC-HRMS for lipidomics analysis.

Sampleprep ID:

SP003198

Sampleprep Summary:

All tissue sample was first homogenized in liquid nitrogen and then 10-20 mg tissues were weighed into a new 1.7 ml Eppendorf tube. Ice cold extraction solvent (400 ul 80% methanol/water) and 10 ul metformin-d6 (50 ng/ul in water) was added to each sample. Geno/Grinder homogenizer was used (1500 rpm, 1 to 2 min) to further break down the tissue chunk and form an even suspension. 200 ul supernatant containing polar metabolites was removed after centrifugation at 20,000 g at 4 °C for 10 min and transferred to LC vial for polar metabolite analysis using LC-HRMS. The rest samples (200 ul solvent and insoluble pellet) were briefly homogenized using Geno/Grinder again (1500 rpm, 30 sec) to loosen the bottom pellet and better mix with extraction solvent. 480 ul MTBE and 10 ul internal standard solution were added. After rigorous vortexing, 120 ul water was added to initiate phase separation. All samples were centrifuged at 20,000 g at 4 °C for 10 min. The supernatant containing lipids was transferred to a new Eppendorf tube and dried using speed vacuum. Dry pellets were stored in -80 °C freezer until ready for LC-HRMS analysis. To extract metabolites and lipids from mouse plasma, 10 ul plasma was mixed with 10 l water containing internal standards (50 ng/ul metformin-d6, 5 mM [U-13C]-glucose and [U-13C]-lactate, and 80 ul ice cold methanol was added. After vortex for 1 min, the mixture was centrifuged with a speed of 20,000 g at 4 °C for 10 min, and 20 ul was transferred directly to LC vial without solvent evaporation, followed by LC-HRMS analysis (injection volume, 3 ul) of polar metabolites such as glucose and metformin. To the rest samples (80 l solvent and pellet), 192 ul MTBE and 10 ul lipid internal standard solution were added. After rigorous vortexing, 48 ul water was added to initiate phase separation. All samples were centrifuged at 20,000 g at 4 °C for 10 min. The supernatant containing lipids was transferred to a new Eppendorf tube and dried using speed vacuum. The dry pellets of lipids from mouse tissues or plasma were reconstituted into (300 ul for tissue and 50 ul for plasma) sample solvent (isopropanol: ethyl acetate, 1:1, v/v), and 3 ul was injected to LC-HRMS for lipidomics analysis.

Combined analysis:

Analysis ID	AN005035
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Xbridge BEH C18 column (100 × 2.1 mm, 2.5 um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Orbitrap Exploris 480
Ion Mode	UNSPECIFIED
Units	arbitrary unit

Chromatography:

Chromatography ID:	CH003806
Instrument Name:	Thermo Vanquish
Column Name:	Xbridge BEH C18 column (100 × 2.1 mm, 2.5 um)
Column Temperature:	40 °C
Flow Gradient:	Linear gradient was: 0 min, 40% B; 1.5 min, 40% B; 5.0 min, 85% B; 12.0 min, 97% B; 16.0 min, 97% B; 16.5 min, 40% B; 21.0 min, 40% B.
Flow Rate:	The flow rate was: 0.15 ml/min.
Solvent A:	80% water/20% acetonitrile; 0.1% formic acid; mM ammonium formate,
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004774
Analysis ID:	AN005035
Instrument Name:	Thermo Orbitrap Exploris 480
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The relevant parameters of Orbitrap Exploris 480 were as listed: vaporizer temperature, 350 °C; ion transfer tube temperature, 300 °C; sheath gas, 35; auxiliary gas, 7; sweep gas, 1; spray voltage, 3.5 kV for positive mode and 2.5 kV for negative mode; RF-lens (%), 45. The resolution was set at 120,000 (at m/z 200). The scan range was 200 to 1600 (m/z). Automatic maximum injection time (max IT) and automatic gain control (AGC) were used. MS/MS scan was acquired using Hybrid mode (BRI-DIA followed by DDA) with the inclusion list containing precursor ions in positive and negative ion mode (described in the Supplementary table 1 of our prior publication PMID: 35842862). The MS/MS condition for positive or negative ion mode was set as follows: precursor isolation window was set at 1 (m/z) for all three methods, HCD collision energy was set at 25%, orbitrap resolution of full scan and MS/MS scan was set at 60,000 and 15,000, respectively. Intensity threshold for DDA and DIA MS/MS scan was set at 10,000. LC-MS peak extraction and integration were performed using MS-DIAL with default settings.
Ion Mode:	UNSPECIFIED