Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA023192

Local Sample ID:	PBS_03
Subject ID:	SU000478
Subject Type:	Animal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammal

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Subject:

Subject ID:	SU000478
Subject Type:	Animal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
PBS_03	SA023192	FL005630	-	Group

Collection:

Collection ID:	CO000472
Collection Summary:	Serum was collected via standard operating procedures.
Sample Type:	Blood

Treatment:

Treatment ID:	TR000492
Treatment Summary:	STAT - low dose antibiotics until week 4 STAT-COHO -STAT treated mice were cohoused with Control mice Control-COHO - Control mice were cohoused with STAT treated mice Control – No antibiotics

Sample Preparation:

Sampleprep ID:	SP000485
Sampleprep Summary:	Sample Preparation Prior to Biocrates p180 Kit Plate Analysis: Thawed serum samples were vortexed for 30 seconds. Whole study pooled QC samples were created by combining 10 µL aliquot from each of the study samples into a 2 mL LoBind eppendorf tube. This QC pooled sample was then vortexed for 30 sec. Then, three whole study pooled QC samples of 30 µL each were aliquoted into 2 mL LoBind eppendorf tubes. Study samples were generated by aliquoting 30 µL from each original sample vial into 2 mL LoBind eppendorf tubes. For extraction, 1,000 µL of cold 3:3:2 Acetonitrile:Isopropyl Alcohol:Water (v/v/v) was added to each tube and vortexed for 5 min at 4 °C. The samples were then centrifuged at 4 °C and at 14,000 rcf for 2 min. A 450 µL aliquot of the supernatant from each sample was transferred into pre-labeled 2.0 mL LoBind eppendorf tubes and stored at -80 °C. Samples were then dried on a lyophilizer overnigh. The residue was reconstituted in 30 µL of 85:15 Ethanol:Water, v/v, and vortexed. Then, the samples were centrifuged at 4 °C for 4 min at 16,000 rcf. Biocrates Plate Preparation: A Biocrates p180 kit was prepared following the AbsoluteIDQ™ p180 Kit metabolomics procedure. Briefly, an internal standard mix was added to 95 of the 96 wells. Next, zero samples, QC standards and calibration standards were added to their corresponding wells. The study samples and pooled QC Samples (20 µL) were then added to the appropriate wells and dried for 30 minutes under nitrogen flow. The plate was derivatized using a 5% phenylisothiocyanate (PITC) solution in (1:1:1) ethanol:pyridine:water (v/v/v) and, then, incubated for 20 minutes followed by a drying step under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all wells. The plate was then shaken and centrifuged. After centrifugation, 150 µL was removed and transferred to a second 96-well plate (LCMS plate). This second plate was diluted with 150 µL of HPLC grade water for a subsequent LCMS (MRM analysis) for measuring amino acids and biogenic amines. All wells in the original plate were diluted with 400 µL of flow injection analysis (FIA) Running Solvent for a FIA-MS (MRM analysis) for measuring lipids, acylcarnitines, and hexose.

Sampleprep ID:

SP000485

Sampleprep Summary:

Sample Preparation Prior to Biocrates p180 Kit Plate Analysis: Thawed serum samples were vortexed for 30 seconds. Whole study pooled QC samples were created by combining 10 µL aliquot from each of the study samples into a 2 mL LoBind eppendorf tube. This QC pooled sample was then vortexed for 30 sec. Then, three whole study pooled QC samples of 30 µL each were aliquoted into 2 mL LoBind eppendorf tubes. Study samples were generated by aliquoting 30 µL from each original sample vial into 2 mL LoBind eppendorf tubes. For extraction, 1,000 µL of cold 3:3:2 Acetonitrile:Isopropyl Alcohol:Water (v/v/v) was added to each tube and vortexed for 5 min at 4 °C. The samples were then centrifuged at 4 °C and at 14,000 rcf for 2 min. A 450 µL aliquot of the supernatant from each sample was transferred into pre-labeled 2.0 mL LoBind eppendorf tubes and stored at -80 °C. Samples were then dried on a lyophilizer overnigh. The residue was reconstituted in 30 µL of 85:15 Ethanol:Water, v/v, and vortexed. Then, the samples were centrifuged at 4 °C for 4 min at 16,000 rcf. Biocrates Plate Preparation: A Biocrates p180 kit was prepared following the AbsoluteIDQ™ p180 Kit metabolomics procedure. Briefly, an internal standard mix was added to 95 of the 96 wells. Next, zero samples, QC standards and calibration standards were added to their corresponding wells. The study samples and pooled QC Samples (20 µL) were then added to the appropriate wells and dried for 30 minutes under nitrogen flow. The plate was derivatized using a 5% phenylisothiocyanate (PITC) solution in (1:1:1) ethanol:pyridine:water (v/v/v) and, then, incubated for 20 minutes followed by a drying step under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all wells. The plate was then shaken and centrifuged. After centrifugation, 150 µL was removed and transferred to a second 96-well plate (LCMS plate). This second plate was diluted with 150 µL of HPLC grade water for a subsequent LCMS (MRM analysis) for measuring amino acids and biogenic amines. All wells in the original plate were diluted with 400 µL of flow injection analysis (FIA) Running Solvent for a FIA-MS (MRM analysis) for measuring lipids, acylcarnitines, and hexose.

Combined analysis:

Analysis ID	AN000716	AN000717	AN000718
Analysis type	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	Reversed phase
Chromatography system	Agilent 1100	Agilent 1100	Agilent 1100
Column	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)
MS Type	ESI	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex API 4000 QTrap	ABI Sciex API 4000 QTrap	ABI Sciex API 4000 QTrap
Ion Mode	POSITIVE	POSITIVE	NEGATIVE
Units	Intensity	Intensity	Intensity

Chromatography:

Chromatography ID:	CH000515
Instrument Name:	Agilent 1100
Column Name:	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS000636
Analysis ID:	AN000716
Instrument Name:	ABI Sciex API 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS000637
Analysis ID:	AN000717
Instrument Name:	ABI Sciex API 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS000638
Analysis ID:	AN000718
Instrument Name:	ABI Sciex API 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	NEGATIVE