Return to study ST000923 main page
MB Sample ID: SA055023
Local Sample ID: | SM-7I9G4 |
Subject ID: | SU000961 |
Subject Type: | Human stool |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU000961 |
Subject Type: | Human stool |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
SM-7I9G4 | SA055023 | FL010109 | UC | Diagnosis |
SM-7I9G4 | SA055023 | FL010109 | Female | sex |
Collection:
Collection ID: | CO000955 |
Collection Summary: | Stool samples were collected by subjects in tubes containing 5 ml of 100% Ethanol and shipped to collection sites. A portion of each stool sample (40-100 mg) and the entire volume of ethanol preservative were stored in 15 mL centrifuge tubes at -80 °C until all samples were collected. |
Sample Type: | Stool |
Additives: | Ethanol |
Treatment:
Treatment ID: | TR000975 |
Treatment Summary: | NA |
Sample Preparation:
Sampleprep ID: | SP000968 |
Sampleprep Summary: | Samples were thawed on ice and then centrifuged (4 ˚C, 5,000 x g) for 5 minutes. Ethanol was evaporated using a gentle stream of nitrogen gas using a nitrogen evaporator (TurboVap LV; Biotage, Charlotte, NC) and stored at -80 ˚C until all samples in the study had been dried. Aqueous homogenates were generated by sonicating each sample in 900 μl of H2O using an ultrasonic probe homogenizer (Branson Sonifier 250) set to a duty cycle of 25% and output control of 2 for 3 minutes. Samples were kept on ice during the homogenization process. The homogenate for each sample was aliquoted into two 10 μL and two 30 μL in 1.5mL centrifuge tubes for LC-MS sample preparation and 30 μL of homogenate from each sample were transferred into a 50 mL conical tube on ice to create a pooled reference sample. The pooled reference mixture was mixed by vortexing and then aliquoted (100 μL per aliquot) into 1.5 mL centrifuge tubes. Aliquots and reference sample aliquots were stored at -80 °C until LC-MS analyses were conducted. Pairs of pooled reference samples were inserted into the queue at intervals of approximately 20 samples in order to assess analytical variance and as a reference to standardize within and across batches by “nearest neighbor” scaling. |
Combined analysis:
Analysis ID | AN001513 | AN001514 | AN001515 | AN001516 |
---|---|---|---|---|
Analysis type | MS | MS | MS | MS |
Chromatography type | HILIC | HILIC | Reversed phase | Reversed phase |
Chromatography system | Shimadzu Nexera X2 | Shimadzu Nexera X2 | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
Column | Atlantis HILIC (Waters; Milford,MA) | Luna NH2 (Phenomenex; Torrance,CA) | Waters ACQUITY UPLC BEH C18 | Waters ACQUITY UPLC BEH C8 (1.7um) |
MS Type | ESI | ESI | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Plus Orbitrap |
Ion Mode | POSITIVE | NEGATIVE | NEGATIVE | POSITIVE |
Units | abundance | abundance | abundance | abundance |
Chromatography:
Chromatography ID: | CH001066 |
Chromatography Summary: | LC-MS samples were prepared from stool homogenates (10 μL) via protein precipitation with the addition of nine volumes 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). The samples were centrifuged (10 min, 9,000 x g, 4°C), and the supernatants injected directly onto a 150 x 2 mm Atlantis HILIC column (Waters; Milford, MA). The column was eluted isocratically at a flow rate of 250 μL/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 1 minute followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes. |
Instrument Name: | Shimadzu Nexera X2 |
Column Name: | Atlantis HILIC (Waters; Milford,MA) |
Flow Gradient: | The column was eluted isocratically with 5% A for 1 minute followed by a linear gradient to 40% B over 10 minutes. |
Flow Rate: | 250 µL/min |
Solvent A: | 100% water; 0.1% formic acid; 10 mM ammonium formate |
Solvent B: | 100% acetonitrile; 0.1% formic acid |
Chromatography Type: | HILIC |
Chromatography ID: | CH001067 |
Chromatography Summary: | LC-MS samples were prepared from stool homogenates (30 μL) via protein precipitation with the addition of four volumes of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). The samples were centrifuged (10 min, 9,000 x g, 4°C) and the supernatants were injected directly onto a 150 x 2.0 mm Luna NH2 column (Phenomenex; Torrance, CA). The column was eluted at a flow rate of 400 μL/min with initial conditions of 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide in water) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol) followed by a 10 min linear gradient to 100% mobile phase A. |
Instrument Name: | Shimadzu Nexera X2 |
Column Name: | Luna NH2 (Phenomenex; Torrance,CA) |
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; mM ammonium hydroxide |
Solvent B: | 75% acetonitrile/25% methanol; 10 mM ammonium hydroxide |
Chromatography Type: | HILIC |
Chromatography ID: | CH001068 |
Chromatography Summary: | Stool homogenates (30 μL) were extracted using 90 μL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) and centrifuged (10 min, 9,000 x g, 4°C). The supernatants (10 μL) were injected onto a 150 x 2.1 mm ACQUITY BEH C18 column (Waters; Milford, MA). The column was eluted isocratically at a flow rate of 450 μL/min with 20% mobile phase A (0.01% formic acid in water) for 3 minutes followed by a linear gradient to 100% mobile phase B (0.01% acetic acid in acetonitrile) over 12 minutes. |
Instrument Name: | Shimadzu Nexera X2 |
Column Name: | Waters ACQUITY UPLC BEH C18 |
Flow Gradient: | The column was eluted isocratically at a flow rate of 450 µL/min with 20% A for 3 minutes followed by a linear gradient to 100% 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 |
Chromatography ID: | CH001069 |
Chromatography Summary: | Lipids (polar and nonpolar) were extracted from stool homogenates (10 μL) using 190 μL of isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL). After centrifugation (10 min, 9,000 x g, ambient temperature), supernatants (10 μL) were injected directly onto a 100 x 2.1 mm ACQUITY BEH C8 column (1.7 μm; Waters; Milford, MA). The column was eluted at a flow rate of 450 μL/min isocratically for 1 minute at 80% mobile phase A (95:5:0.1 vol/vol/vol 10 mM ammonium acetate/methanol/acetic acid), followed by a linear gradient to 80% mobile-phase B (99.9:0.1 vol/vol methanol/acetic acid) over 2 minutes, a linear gradient to 100% mobile phase B over 7 minutes, and then 3 minutes at 100% mobile-phase B. |
Instrument Name: | Shimadzu Nexera X2 |
Column Name: | Waters ACQUITY UPLC BEH C8 (1.7um) |
Flow Gradient: | The column was eluted 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; 0.1% acetic acid; 10 mM ammonium acetate |
Solvent B: | 100% methanol; 0.1% acetic acid |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS001396 |
Analysis ID: | AN001513 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
Ion Mode: | POSITIVE |
Scanning Range: | 70-800 |
MS ID: | MS001397 |
Analysis ID: | AN001514 |
Instrument Name: | Thermo Q Exactive Plus Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
Ion Mode: | NEGATIVE |
Scanning Range: | 60-750 |
MS ID: | MS001398 |
Analysis ID: | AN001515 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
Ion Mode: | NEGATIVE |
Scanning Range: | 70-850 |
MS ID: | MS001399 |
Analysis ID: | AN001516 |
Instrument Name: | Thermo Q Exactive Plus Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
Ion Mode: | POSITIVE |
Scanning Range: | 200-1100 |