Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA207401

Local Sample ID:	HFD-PV_30_12121
Subject ID:	SU002247
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002247
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
HFD-PV_30_12121	SA207401	FL025425	Wild-type	Genotype
HFD-PV_30_12121	SA207401	FL025425	HFD	Diet
HFD-PV_30_12121	SA207401	FL025425	3 days	Diet Duration
HFD-PV_30_12121	SA207401	FL025425	Control	Treatment
HFD-PV_30_12121	SA207401	FL025425	Portal Vein Serum	Tissue
HFD-PV_30_12121	SA207401	FL025425	30	Collection Time (minute)
HFD-PV_30_12121	SA207401	FL025425	Water	Drinking

Collection:

Collection ID:	CO002240
Collection Summary:	After 3 days or 4 weeks of either HFD or CD feeding, each mouse was administrated a 1g/kg U-13C fructose oral gavage. Mice were anesthetized with 2% isoflurane, and their abdominal cavities were opened at the specified time. Abdominal tissues were displaced to identify the portal vein, and a 27G needle was used to immediately collect portal vein blood samples (~100 µL). Immediately after, cardiac blood was collected from the right ventricle using a 25G needle (~1 mL). The jejunum, pancreas, and liver tissues were quickly resected and snap-frozen in liquid nitrogen. Blood samples were placed on ice in the absence of anticoagulant for 20 minutes and centrifuged at 16,000 × g for 10 minutes at 4 °C to separate the serum and plasma fractions of each sample. Serum and tissue samples were kept at −80 °C until further analysis.
Collection Protocol Filename:	cwwong_methods.pdf
Sample Type:	Tissue

Treatment:

Treatment ID:	TR002259
Treatment Summary:	After 3 days or 4 weeks of either HFD or CD feeding, each mouse was administrated a 1g/kg U-13C fructose oral gavage. For ad-lib fructose feeding experiments, mice were fed fructose in the drinking water (15% w/v) for 4 weeks. Saline, fructose and glycerate injection were done in CD-fed mice for 12 weeks. For glycerate kinetic study, glycerate injections were performed in treatment-navie mice.

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.

Combined analysis:

Analysis ID	AN003540	AN003541
Analysis type	MS	MS
Chromatography type	HILIC	IR-MALDI
Chromatography system	Thermo Vanquish	Other
Column	Waters XBridge BEH Amide (150mm x 2.1mm,2.5um)	Other
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap	Thermo Exploris 240 Orbitrap
Ion Mode	NEGATIVE	NEGATIVE
Units	Total Abundance	Peak Area

Chromatography:

Chromatography ID:	CH002614
Instrument Name:	Thermo Vanquish
Column Name:	Waters XBridge BEH Amide (150mm x 2.1mm,2.5um)
Chromatography Type:	HILIC

Chromatography ID:	CH002615
Instrument Name:	Other
Column Name:	Other
Chromatography Type:	IR-MALDI

MS:

MS ID:	MS003298
Analysis ID:	AN003540
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Fullscan mass spectrometry. The full scan mass spectrometry analysis was performed on a Thermo Q Exactive PLUS with a HESI source which was set to a spray voltage of -2.7kV under negative mode and 3.5kV under positive mode. The sheath, auxiliary, and sweep gas flow rates of 40, 10, and 2 (arbitrary unit), respectively. The capillary temperature was set to 300°C, and the aux gas heater was 360°C. The S-lens RF level was 45. The m/z range was set to 72 to 1000 m/z under both positive and negative ionization mode. The AGC target was set to 3e6, and the maximum IT was 200 ms. The mass resolution (full-width half maximum) was set to 70,000 @ m/z = 200.
Ion Mode:	NEGATIVE
Analysis Protocol File:	cwwong_methods.pdf

MS ID:	MS003299
Analysis ID:	AN003541
Instrument Name:	Thermo Exploris 240 Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	To ablate targeted tissue regions a 2970-nm wavelength laser was used with a single burst of ten pulses to produce 1 mJ of energy at a rate of 10 kHz. X and Y stage movements of 100 µm were used to achieve oversampling(Nazari and Muddiman, 2015). Ablated analytes were post-ionized in the orthogonal electrospray plume, established by applying a voltage of approximately 3 kV to the electrospray solvent (1 mM acetic acid in 50:50 water/acetonitrile.) Mass spectrometry analysis of ionized molecules was performed in negative mode with internal calibrant used to achieve high mass accuracy (<2.5 ppm) within the 85-225 m/z range. Automatic gain control (AGC) was disabled. A mass resolution power of 240,000FWHM at 200 m/z was used with a fixed injection time (15 ms) to synchronize timing of the ablation plume with ion collection in the C-trap of the mass spectrometer.
Ion Mode:	NEGATIVE
Analysis Protocol File:	cwwong_methods.pdf