Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA247618

Local Sample ID:	STL10279
Subject ID:	SU002561
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002561
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
STL10279	SA247618	FL031125	0	collectionWeek
STL10279	SA247618	FL031125	mild	PUCAI_C3_WKall

Collection:

Collection ID:	CO002554
Collection Summary:	Pediatric UC patients (4-17 years of age) were recruited between July 2012 and April 2015 from 29 centers in the USA and Canada and monitored over the course of one year. Patients were treatment-naive at week 0 and received either 5-aminosalicylic acid (mesalamine) or oral/intravenous corticosteroids followed by mesalamine. Up to 4 samples were collected from each patient (week 0, 4, 12 and 52). In addition, metadata and clinical data were collected, including age, gender, ethnicity, treatment, Pediatric Ulcerative Colitis Activity Index (PUCAI), disease progression (colectomy and remission status) and fecal calprotectin (ELISA). Stool samples were frozen at -20 degrees at time of collection by study participants and transported back to the site via freezer gel packs and shipping containers to keep samples frozen during transport. Once at the site, samples were frozen at -80 degrees and batched-shipped with dry ice overnight to the biorepository.
Sample Type:	Feces

Treatment:

Treatment ID:	TR002573
Treatment Summary:	NA

Sample Preparation:

Sampleprep ID:	SP002567
Sampleprep Summary:	Stool samples were homogenized in 10 μL of water per milligram stool sample weight using a bead mill (TissueLyser II; Qiagen) and the aqueous homogenates were aliquoted for metabolite profiling analyses. LC-MS samples were prepared for four profiling methods as follows: HILIC-pos: Metabolites were extracted by adding 90 μL of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C8-pos: Lipids were extracted by adding 190 μL of isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, ambient temperature) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. HILIC-neg: Metabolites were extracted by adding 120 μL of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C18-neg: Metabolites were extracted by adding 90 μL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis.

Sampleprep ID:

SP002567

Sampleprep Summary:

Stool samples were homogenized in 10 μL of water per milligram stool sample weight using a bead mill (TissueLyser II; Qiagen) and the aqueous homogenates were aliquoted for metabolite profiling analyses. LC-MS samples were prepared for four profiling methods as follows: HILIC-pos: Metabolites were extracted by adding 90 μL of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C8-pos: Lipids were extracted by adding 190 μL of isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, ambient temperature) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. HILIC-neg: Metabolites were extracted by adding 120 μL of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C18-neg: Metabolites were extracted by adding 90 μL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis.

Combined analysis:

Analysis ID	AN004033	AN004034	AN004035	AN004036
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	Reversed phase	HILIC	Reversed phase
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters Atlantis HILIC (150 x 2 mm, 3 µm)	Waters Acquity BEH C8 (100 x 2.1mm, 1.7um)	Phenomenex Luna NH2 (150 x 2.1mm, 3um)	Waters ACQUITY UPLC BEH C18 (150 x 1.7mm,2.1um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	POSITIVE	NEGATIVE	NEGATIVE
Units	Abundance	Abundance	Abundance	Abundance

Chromatography:

Chromatography ID:	CH002981
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Atlantis HILIC (150 x 2 mm, 3 µm)
Column Temperature:	30C
Flow Gradient:	Isocratically with 5% mobile phase A for 1 minute followed by a linear gradient to 40% mobile phase B over 10 minutes
Flow Rate:	250 µL/min
Solvent A:	100% water; 10 mM ammonium formate; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH002982
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Acquity BEH C8 (100 x 2.1mm, 1.7um)
Column Temperature:	40C
Flow Gradient:	The column was eluted at a flow rate of 450 µL/min isocratically for 1 minute at 80% mobile phase A, followed by a linear gradient to 80% mobile-phase B over 2 minutes, a linear gradient to 100% mobile phase B over 7 minutes, and then 3 minutes at 100% mobile-phase B.
Flow Rate:	450 µL/min
Solvent A:	95% water/5% methanol; 10 mM ammonium acetate; 0.1% acetic acid
Solvent B:	100% methanol; 0.1% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH002983
Instrument Name:	Shimadzu Nexera X2
Column Name:	Phenomenex Luna NH2 (150 x 2.1mm, 3um)
Column Temperature:	30C
Flow Gradient:	The column was eluted with initial conditions of 10% mobile phase A and 90% mobile phase B followed by a 10 min linear gradient to 100% mobile phase A.
Flow Rate:	400 µL/min
Solvent A:	100% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide
Solvent B:	75% acetonitrile/25% methanol; 10 mM ammonium hydroxide
Chromatography Type:	HILIC

Chromatography ID:	CH002984
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters ACQUITY UPLC BEH C18 (150 x 1.7mm,2.1um)
Column Temperature:	45C
Flow Gradient:	The column was eluted isocratically at a flow rate of 450 µL/min with 20% mobile phase A for 3 minutes followed by a linear gradient to 100% mobile phase B over 12 minutes.
Flow Rate:	450 µL/min
Solvent A:	100% water; 0.01% formic acid
Solvent B:	100% acetonitrile; 0.01% acetic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003780
Analysis ID:	AN004033
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples.
Ion Mode:	POSITIVE

MS ID:	MS003781
Analysis ID:	AN004034
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples.
Ion Mode:	POSITIVE

MS ID:	MS003782
Analysis ID:	AN004035
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples.
Ion Mode:	NEGATIVE

MS ID:	MS003783
Analysis ID:	AN004036
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples.
Ion Mode:	NEGATIVE