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MB Sample ID: SA227293
| Local Sample ID: | GU2 |
| Subject ID: | SU002406 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 5-77 |
| Weight Or Weight Range: | NA |
| Height Or Height Range: | NA |
| Gender: | Male and female |
| Human Lifestyle Factors: | Urban industrial; Rural industrial; Rural traditional; Isolated traditional |
| Human Alcohol Drug Use: | NA |
| Human Nutrition: | NA |
| Human Inclusion Criteria: | NA |
| Human Exclusion Criteria: | NA |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002406 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 5-77 |
| Weight Or Weight Range: | NA |
| Height Or Height Range: | NA |
| Gender: | Male and female |
| Human Lifestyle Factors: | Urban industrial; Rural industrial; Rural traditional; Isolated traditional |
| Human Alcohol Drug Use: | NA |
| Human Nutrition: | NA |
| Human Inclusion Criteria: | NA |
| Human Exclusion Criteria: | NA |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| GU2 | SA227293 | FL027609 | Guayabo | Population |
| GU2 | SA227293 | FL027609 | Rural Industrial | Industrialization_Category |
| GU2 | SA227293 | FL027609 | F | Sex |
| GU2 | SA227293 | FL027609 | 19 | Age |
| GU2 | SA227293 | FL027609 | Within 4 days | Time_Kept_on_Ice_before_Frozen |
Collection:
| Collection ID: | CO002399 |
| Collection Summary: | Fecal material was deposited into polypropylene containers and then put on ice. Samples were kept in ice while in the field until arriving at research facilities equipped with freezers. The Norman samples were kept in ice after collection and frozen at the laboratory within 24 hours. The Peruvian samples were secured similarly to the Norman samples. After collection, samples were stored on ice for four days until arriving at Lima, Peru. Samples were frozen and sent to the laboratory in Norman, Oklahoma. Boulkiemdé samples were collected similarly to Norman and Peruvian samples. After collection, Boulkiemdé samples were frozen at -20 °C within 24 hours and kept frozen overnight. Samples were thawed the following evening to extract DNA, refrozen at -20 °C, and kept frozen until shipped to the laboratory in Norman, Oklahoma. Upon arrival, 2 g of fecal material was extracted from each sample for anaerobic culturing. Following this 2 g aliquoting, samples were frozen at -80 °C until use for this project. The Norman, Tunapuco, and Matses samples had previously been aliquoted and underwent 16S rRNA gene sequencing for an earlier study. |
| Sample Type: | Feces |
| Collection Location: | United States; Peru; Burkina Faso |
| Volumeoramount Collected: | Variable; 50mg used for experimental analyses |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002418 |
| Treatment Summary: | The sample preparation protocol used for this project was adapted from a global metabolite extraction protocol with proven success (Want et al. 2013). Samples were thawed and 500 μl of chilled LC-MS grade water (Fisher Scientific) was added to 50 mg of fecal material. Next, a TissueLyzer homogenized samples at 25 Hz for three minutes. Following homogenization, chilled LC-grade methanol (Fisher Scientific) spiked with 4 μM sulfachloropyridazine as the internal standard (IS) was added, bringing the total concentration to 50% methanol. The TissueLyzer homogenized samples again at 25 Hz for three minutes, followed by overnight incubation at 4 °C. The next day, samples were centrifuged at 16,000 x g at 4 °C for ten minutes. Aqueous supernatant was then removed and dried using a SpeedVac vacuum concentrator. Dried extracts were frozen at -80 °C until the day of MS analysis. Immediately prior to MS analysis, extracts were resuspended in 150 μl chilled LC-MS methanol:water (1:1) spiked with 1 μg/ml sulfadimethoxine as a second IS. After resuspension, samples were diluted to a 1:10 ratio. Diluted samples were sonicated using a Fisher Scientific Ultrasonic Cleaning Bath at maximum power for ten minutes. Supernatants were spun briefly to remove any particulates, then loaded into a 96-well plate for MS analysis. One well contained only 150 μl of the resuspension solution to serve as blank control. |
Sample Preparation:
| Sampleprep ID: | SP002412 |
| Sampleprep Summary: | The sample preparation protocol used for this project was adapted from a global metabolite extraction protocol with proven success (Want et al. 2013). Samples were thawed and 500 μl of chilled LC-MS grade water (Fisher Scientific) was added to 50 mg of fecal material. Next, a TissueLyzer homogenized samples at 25 Hz for three minutes. Following homogenization, chilled LC-grade methanol (Fisher Scientific) spiked with 4 μM sulfachloropyridazine as the internal standard (IS) was added, bringing the total concentration to 50% methanol. The TissueLyzer homogenized samples again at 25 Hz for three minutes, followed by overnight incubation at 4 °C. The next day, samples were centrifuged at 16,000 x g at 4 °C for ten minutes. Aqueous supernatant was then removed and dried using a SpeedVac vacuum concentrator. Dried extracts were frozen at -80 °C until the day of MS analysis. Immediately prior to MS analysis, extracts were resuspended in 150 μl chilled LC-MS methanol:water (1:1) spiked with 1 μg/ml sulfadimethoxine as a second IS. After resuspension, samples were diluted to a 1:10 ratio. Diluted samples were sonicated using a Fisher Scientific Ultrasonic Cleaning Bath at maximum power for ten minutes. Supernatants were spun briefly to remove any particulates, then loaded into a 96-well plate for MS analysis. One well contained only 150 μl of the resuspension solution to serve as blank control. |
| Extract Storage: | On ice |
Combined analysis:
| Analysis ID | AN003787 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | ThermoFisher Scientific Vanquish Flex Binary LC System |
| Column | Kinetex C18 core-shell (50 x 2.1mm,1.7um,100Å) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH002801 |
| Methods Filename: | C18_RP_Pos_QEPlus_20180403_12.5mint_ddMS2_Chrom.txt |
| Instrument Name: | ThermoFisher Scientific Vanquish Flex Binary LC System |
| Column Name: | Kinetex C18 core-shell (50 x 2.1mm,1.7um,100Å) |
| Column Temperature: | 40 |
| Flow Gradient: | 12.5 min runtime gradient |
| Internal Standard: | Sulfadimethoxine |
| Sample Injection: | 5 uL randomized order |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003530 |
| Analysis ID: | AN003787 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | LC was performed on a ThermoFisher Scientific Vanquish Flex Binary LC System with a Kinetex C18 core-shell column (50 x 2.1 mm, 1.7 μM particle size, 100 Å pore size). LC column was kept at 40 °C and the sample compartment was held at 10 °C. The LC System was coupled to a ThermoFisher Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer for MS/MS analysis. For the LC mobile phase, Solvent A was LC-MS grade water (Fisher Scientific) with 0.1% formic acid and Solvent B was LC-MS grade acetonitrile (Fisher Scientific) with 0.1% formic acid. Elution gradient started at 5% Solvent B for one minute, increased to 100% Solvent B until minute nine, held at 100% Solvent B for two minutes, dropped to 5% Solvent B over 30 seconds, and 5% Solvent B for one minute as re-equilibration. Samples were injected in random order with an injection volume of 5 μl. After elution, electrospray ionization was conducted with spray voltage of 3.8 kV, auxiliary gas flow rate of 10, auxiliary gas temperature at 350 °C, sheath gas flow rate at 35, and sweep gas flow at 0. Capillary temperature was 320 °C and S-lens RF was 50 V. MS1 scan range was 100-1,500 m/z, MS1 resolution was set to 35,000 and MS1 AGC target to 1e6. MS1 data were obtained in positive mode and MS2 data were obtained using data-dependent acquisition. In each cycle, 5 MS/MS scans of the most abundant ion were recorded. Both MS1 and MS2 injection times were set at 100 ms. MS2 resolutions were set to 17,500, MS2 AGC target was set to 5e5, and the inclusion window to 2 m/z. MS/MS was conducted at an apex trigger of 2-8 seconds and an exclusion window of 10 seconds. MS/MS collision energy gradually increased from 20-40%. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 320°C |
| Ionization: | ESI |
| Analysis Protocol File: | C18_RP_Pos_QEPlus_20180403_12.5mint_ddMS2_MS.txt |
