Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA251538

Local Sample ID:	59
Subject ID:	SU002601
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU002601
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
59	SA251538	FL032043	Inulin	Fiber
59	SA251538	FL032043	30	Timepoint
59	SA251538	FL032043	F	Sex

Collection:

Collection ID:	CO002594
Collection Summary:	The study is a longitudinal, randomized crossover design in which 18 consented participants (8 men and 10 women) had their diets periodically supplemented with two fibers, arabinoxylan (AX) and long-chain inulin, and a mixture of fibers consisting of equal parts AX, LCI, acacia gum, glucomannans, and resistant starch. Participants were randomized to consume either AX or LCI first, and the mixed fibers were always administered last. For each of the fiber cycles, blood, urine and stool samples were collected at seven timepoints: baseline, end of week one, end of week two, end of week three, day 3 after end of supplementation and day 10 after end of supplementation. Blood was fractionated into plasma, serum and peripheral blood mononucleotide cells (PBMCs). Metabolomics was performed on the plasma fraction of the blood. Once all samples were in the correct aliquots, they were stored at -80C. Samples were only thawed when prepared for analysis for each of the respective omics assay which were all performed within 5 years of collecting the samples.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR002613
Treatment Summary:	Participants went through 3 cycles of fiber supplementation, each cycle was three weeks long with weekly increasing doses of 10 g/day during the first week, 20g/day during the second week and 30 g/day during the third week. Randomized for the first two cycles, fibers tested were chicory inulin (99%>5 dp; range 2-60dp; average >23 dp) and arabinoxylan (psyllium husks powder Now Foods) and a mix of 5 fibers during the third cycle. The fiber mix included equal amounts of inulin, arabinoxylan, glucomannan (Now Foods), resistant starch (Hi Maize from Honeyville), and acacia fiber (Now Foods). Washout period between the cycles was from 6-10 weeks. Fiber was provided in 10 g sachets and participants were instructed to resuspend content of the sachet in at least 8 oz of water and drink one with breakfast for the first week, one with breakfast and one with dinner during the second week, and one with each meal (breakfast, lunch and dinner) during the third week.

Treatment ID:

TR002613

Treatment Summary:

Participants went through 3 cycles of fiber supplementation, each cycle was three weeks long with weekly increasing doses of 10 g/day during the first week, 20g/day during the second week and 30 g/day during the third week. Randomized for the first two cycles, fibers tested were chicory inulin (99%>5 dp; range 2-60dp; average >23 dp) and arabinoxylan (psyllium husks powder Now Foods) and a mix of 5 fibers during the third cycle. The fiber mix included equal amounts of inulin, arabinoxylan, glucomannan (Now Foods), resistant starch (Hi Maize from Honeyville), and acacia fiber (Now Foods). Washout period between the cycles was from 6-10 weeks. Fiber was provided in 10 g sachets and participants were instructed to resuspend content of the sachet in at least 8 oz of water and drink one with breakfast for the first week, one with breakfast and one with dinner during the second week, and one with each meal (breakfast, lunch and dinner) during the third week.

Sample Preparation:

Sampleprep ID:	SP002607
Sampleprep Summary:	We performed an untargeted metabolomics using a platform, previously described in Contrepois et al., 2015 (Contrepois et al., 2015) to profile plasma extracted metabolites. Samples were thawed and 100 μL of plasma was transferred into a 2mL Protein Lobind Eppendorf tube. In order to precipitate and remove proteins, 400 μL of 1:1:1 methanol:acetonitrile:acetone solution, including 17 internal standards to control for extraction efficiency and evaluate LC-MS performance, was added to each plasma sample while on a cold plate. Samples were placed on the vortex shaker for 15 minutes at 4C then placed at -20C for two hours to allow for sufficient protein precipitation. Samples were centrifuged at 10,000 rpm for 10 min at 4C and the supernatant (metabolite extract) from each sample was carefully removed and placed in a new 2mL Protein Lobind Eppendorf tube. Samples were placed in the turbovap, evaporated to dryness under nitrogen and stored at -80C. Before analysis on the MS, samples were reconstituted in 100 μL 1:1 methanol:water solution and vortexed for 30s, placed in a bath sonicator for 30s and incubated on ice for 30s. This process was repeated 3 times for all samples and were centrifuged at 10,000 rpm for 10 min at 4C. The supernatant of each of these was then transferred to MS tubes and stored at -20C until ran on the mass spectrometer.

Sampleprep ID:

SP002607

Sampleprep Summary:

We performed an untargeted metabolomics using a platform, previously described in Contrepois et al., 2015 (Contrepois et al., 2015) to profile plasma extracted metabolites. Samples were thawed and 100 μL of plasma was transferred into a 2mL Protein Lobind Eppendorf tube. In order to precipitate and remove proteins, 400 μL of 1:1:1 methanol:acetonitrile:acetone solution, including 17 internal standards to control for extraction efficiency and evaluate LC-MS performance, was added to each plasma sample while on a cold plate. Samples were placed on the vortex shaker for 15 minutes at 4C then placed at -20C for two hours to allow for sufficient protein precipitation. Samples were centrifuged at 10,000 rpm for 10 min at 4C and the supernatant (metabolite extract) from each sample was carefully removed and placed in a new 2mL Protein Lobind Eppendorf tube. Samples were placed in the turbovap, evaporated to dryness under nitrogen and stored at -80C. Before analysis on the MS, samples were reconstituted in 100 μL 1:1 methanol:water solution and vortexed for 30s, placed in a bath sonicator for 30s and incubated on ice for 30s. This process was repeated 3 times for all samples and were centrifuged at 10,000 rpm for 10 min at 4C. The supernatant of each of these was then transferred to MS tubes and stored at -20C until ran on the mass spectrometer.

Combined analysis:

Analysis ID	AN004115	AN004116	AN004117	AN004118
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS
Column	SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)	SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)	Agilent Zorbax SBaq (2.1 x 50 mm, 1.8 um)	Agilent Zorbax SBaq (2.1 x 50 mm, 1.8 um)
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 Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Area	Area	Area	Area

Chromatography:

Chromatography ID:	CH003049
Chromatography Summary:	HILIC experiments were performed using a ZIC-HILIC column 2.1x100 mm, 3.5μm, 200Å (Merck Millipore) and mobile phase solvents consisting of 10mM ammonium acetate in 50/50 acetonitrile/water (A) and 10 mM ammonium acetate in 95/5 acetonitrile/water (B).(Contrepois et al., 2015)
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)
Column Temperature:	40
Flow Gradient:	The gradient profile used was 99%B for 2 minutes, 99-1% B over 15 minutes, 1%B for 3 minutes. 99%B for 5 minutes.
Flow Rate:	.5 ml/min
Solvent A:	10mM ammonium acetate in 50/50 acetonitrile/water
Solvent B:	10 mM ammonium acetate in 95/5 acetonitrile/water
Chromatography Type:	HILIC

Chromatography ID:	CH003050
Chromatography Summary:	RPLC experiments were performed using a Zorbax SBaq column 2.1 x 50 mm, 1.8 μm, 100Å (Agilent Technologies) and mobile phase solvents consisting of 0.06% acetic acid in water (A) and 0.06% acetic acid in methanol (B). (Contrepois et al., 2015)
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Agilent Zorbax SBaq (2.1 x 50 mm, 1.8 um)
Column Temperature:	60
Flow Gradient:	The gradient profile used was 1% B for 1 minute, 1%-80% B over 8 minutes, 80% B for 3 minutes, 1% B for 5 minutes.
Flow Rate:	.6ml/min
Solvent A:	0.06% acetic acid in water
Solvent B:	0.06% acetic acid in methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003862
Analysis ID:	AN004115
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were imported into Progenesis QI 2.3 software (Water, Milford, MA, USA) to align and quantify chromatographic peaks. Data from all 4 acquisition modes (HILIC positive, HILIC negative, RPLC positive, RPLC negative) were processed independently. Using in house R code, we 1) removed noise, 2) imputed data and 3) adjusted for MS drift with time using the LOESS normalization method on pooled QCs injected every 10 injections in the sequence. We used MetID and our MS/MS data to identify 12740 metabolites with confidence levels ranging from 1-3, where 1 matches MS/MS, retention time and m/z from standards on our platform (843 metabolites), 2 has MS/MS and m/z matches from a database (395 metabolites), and 3 matches the m/z of a database (11,502).
Ion Mode:	POSITIVE

MS ID:	MS003863
Analysis ID:	AN004116
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were imported into Progenesis QI 2.3 software (Water, Milford, MA, USA) to align and quantify chromatographic peaks. Data from all 4 acquisition modes (HILIC positive, HILIC negative, RPLC positive, RPLC negative) were processed independently. Using in house R code, we 1) removed noise, 2) imputed data and 3) adjusted for MS drift with time using the LOESS normalization method on pooled QCs injected every 10 injections in the sequence. We used MetID and our MS/MS data to identify 12740 metabolites with confidence levels ranging from 1-3, where 1 matches MS/MS, retention time and m/z from standards on our platform (843 metabolites), 2 has MS/MS and m/z matches from a database (395 metabolites), and 3 matches the m/z of a database (11,502).
Ion Mode:	NEGATIVE

MS ID:	MS003864
Analysis ID:	AN004117
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were imported into Progenesis QI 2.3 software (Water, Milford, MA, USA) to align and quantify chromatographic peaks. Data from all 4 acquisition modes (HILIC positive, HILIC negative, RPLC positive, RPLC negative) were processed independently. Using in house R code, we 1) removed noise, 2) imputed data and 3) adjusted for MS drift with time using the LOESS normalization method on pooled QCs injected every 10 injections in the sequence. We used MetID and our MS/MS data to identify 12740 metabolites with confidence levels ranging from 1-3, where 1 matches MS/MS, retention time and m/z from standards on our platform (843 metabolites), 2 has MS/MS and m/z matches from a database (395 metabolites), and 3 matches the m/z of a database (11,502).
Ion Mode:	POSITIVE

MS ID:	MS003865
Analysis ID:	AN004118
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were imported into Progenesis QI 2.3 software (Water, Milford, MA, USA) to align and quantify chromatographic peaks. Data from all 4 acquisition modes (HILIC positive, HILIC negative, RPLC positive, RPLC negative) were processed independently. Using in house R code, we 1) removed noise, 2) imputed data and 3) adjusted for MS drift with time using the LOESS normalization method on pooled QCs injected every 10 injections in the sequence. We used MetID and our MS/MS data to identify 12740 metabolites with confidence levels ranging from 1-3, where 1 matches MS/MS, retention time and m/z from standards on our platform (843 metabolites), 2 has MS/MS and m/z matches from a database (395 metabolites), and 3 matches the m/z of a database (11,502).
Ion Mode:	NEGATIVE