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MB Sample ID: SA251638
Local Sample ID: | 323 |
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 |
---|---|---|---|---|
323 | SA251638 | FL032055 | Mix | Fiber |
323 | SA251638 | FL032055 | 20 | Timepoint |
323 | SA251638 | FL032055 | 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. |
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. |
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 |