Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA207440

Local Sample ID:	HFD-HV_45_12122
Subject ID:	SU002247
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Sample Preparation:

Sampleprep ID:	SP002253
Sampleprep Summary:	For LC-MS: Metabolites were extracted using a protocol optimized for water-soluble polar metabolite analysis using liquid chromatography coupled with mass spectrometry. All extraction buffers were stored at -20°C prior to usage and immediately preceding the metabolite extraction. For serum samples, a 10 μL serum aliquot was used for fructose tracing. Metabolites were extracted with 40 μL of ice-cold methanol and incubated at -20 °C for 20 minutes. The clean supernatant was collected after centrifugation for 10 minutes at the highest speed, and the leftover pellet was further treated with 200 µL cold extraction buffer (40:40:20 v/v/v methanol:acetonitrile:water solution) and left to incubate on crushed ice for an additional 10 minutes. Following an additional 10 minutes of centrifugation at the highest speed, the clean supernatant was collected and pooled with the supernatant from the first collection. For tissue samples, the extraction buffer used was a (v/v/v) solution of 40:40:20 (methanol:acetonitrile:water) + 0.1 M formic acid. An aliquot volume equivalent to 20x the sample weight was added to the Eppendorf tube with the homogenized sample, vortexed for 10 seconds, and left to chill on crushed ice for 10 minutes. The samples were then centrifuged for 10 minutes at the highest speed at 4°C. The supernatant was transferred to a correspondingly labeled and chilled Eppendorf tube, and the process was repeated once more. The total volume of supernatant was then centrifuged for an additional 10 minutes. After centrifugation, a final 500µL aliquot of the homogenate was then pipetted to a second clean Eppendorf tube, to which 44µL of 15% (m/v) NH4HCO3 was added to neutralize the acid in the buffer and precipitate the protein. This is the final sample extract to be vialed and loaded to the instrument for analysis. Metabolite extracts were stored at -80 °C until analysis. For MSI: Pancreas tissues were first equilibrated to -15°C then sectioned to 20 µm thickness using a Leica CM1950 cryostat (Buffalo Grove, IL, USA). Cut sections were then thaw-mounted on clean microscope slides (1 mm height, plain, Fisher Scientific, Pittsburgh, PA) and stored at -80 °C until IR-MALDESI-MSI analysis. Tissues were first blocked into four groups, each containing one replicate of each condition. Tissues were both cut and imaged in randomized order within these blocks to minimize sampling bias.

Sampleprep ID:

SP002253

Sampleprep Summary:

For LC-MS: Metabolites were extracted using a protocol optimized for water-soluble polar metabolite analysis using liquid chromatography coupled with mass spectrometry. All extraction buffers were stored at -20°C prior to usage and immediately preceding the metabolite extraction. For serum samples, a 10 μL serum aliquot was used for fructose tracing. Metabolites were extracted with 40 μL of ice-cold methanol and incubated at -20 °C for 20 minutes. The clean supernatant was collected after centrifugation for 10 minutes at the highest speed, and the leftover pellet was further treated with 200 µL cold extraction buffer (40:40:20 v/v/v methanol:acetonitrile:water solution) and left to incubate on crushed ice for an additional 10 minutes. Following an additional 10 minutes of centrifugation at the highest speed, the clean supernatant was collected and pooled with the supernatant from the first collection. For tissue samples, the extraction buffer used was a (v/v/v) solution of 40:40:20 (methanol:acetonitrile:water) + 0.1 M formic acid. An aliquot volume equivalent to 20x the sample weight was added to the Eppendorf tube with the homogenized sample, vortexed for 10 seconds, and left to chill on crushed ice for 10 minutes. The samples were then centrifuged for 10 minutes at the highest speed at 4°C. The supernatant was transferred to a correspondingly labeled and chilled Eppendorf tube, and the process was repeated once more. The total volume of supernatant was then centrifuged for an additional 10 minutes. After centrifugation, a final 500µL aliquot of the homogenate was then pipetted to a second clean Eppendorf tube, to which 44µL of 15% (m/v) NH4HCO3 was added to neutralize the acid in the buffer and precipitate the protein. This is the final sample extract to be vialed and loaded to the instrument for analysis. Metabolite extracts were stored at -80 °C until analysis. For MSI: Pancreas tissues were first equilibrated to -15°C then sectioned to 20 µm thickness using a Leica CM1950 cryostat (Buffalo Grove, IL, USA). Cut sections were then thaw-mounted on clean microscope slides (1 mm height, plain, Fisher Scientific, Pittsburgh, PA) and stored at -80 °C until IR-MALDESI-MSI analysis. Tissues were first blocked into four groups, each containing one replicate of each condition. Tissues were both cut and imaged in randomized order within these blocks to minimize sampling bias.