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MB Sample ID: SA074295
| Local Sample ID: | 40023454_2 |
| Subject ID: | SU001135 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 25-78 yr |
| Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001135 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 25-78 yr |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 40023454_2 | SA074295 | FL011438 | PLACEBO | Aspirin Dosage |
| 40023454_2 | SA074295 | FL011438 | Colon Mucosal Tissue | Sample.Matrix |
Collection:
| Collection ID: | CO001129 |
| Collection Summary: | Fresh frozen normal colon mucosal tissue samples were collected from participants in the Aspirin/Folate Polyp Prevention Study [1, 2] at the year three (end of treatment) colonoscopy. After the endoscope was advanced to the cecum, biopsy specimens were obtained from the mid-ascending colon (5 cm above the ileocecal valve). Immediately after the biopsies were taken, they were removed from the forceps, placed into 1.8 ml freezer tubes and immersed in liquid nitrogen or a dry ice/ethanol slurry until storage in a -70C freezer. The specimens were shipped on dry ice from the clinical centers to the Dartmouth biorepository storage facility and subsequently to the metabolomics analysis lab at Emory University. In addition, a colon tissue reference was generated using normal colon tissue removed at surgery from an anonymous donor. References 1. Baron JA, Cole BF, Sandler RS, et al. A randomized trial of aspirin to prevent colorectal adenomas. The New England journal of medicine. 2003;348(10):891-9. 2. Cole BF, Baron JA, Sandler RS, et al. Folic acid for the prevention of colorectal adenomas: a randomized clinical trial. JAMA : the journal of the American Medical Association. 2007;297(21):2351-9. |
| Sample Type: | Colon mucosal tissue |
| Storage Conditions: | Described in summary |
Treatment:
| Treatment ID: | TR001149 |
| Treatment Summary: | Samples were received frozen in aliquouts of <250uL. Prior to analysis, samples were thawed and prepared for HRM analysis using the standard protocols described in the Sample Preparation section. |
Sample Preparation:
| Sampleprep ID: | SP001142 |
| Sampleprep Summary: | Samples were prepared for metabolomics analysis using established methods(Johnson et al. (2010). Analyst; Go et al. (2015). Tox Sci). Prior to analysis, plasma aliquots were removed from storage at -80 degrees C and thawed on ice. Each cryotube was then vortexed briefly to ensure homogeneity, and 50 microliters was transferred to a clean microfuge tube. Immediately after, the samples were treated with 200 microliters of ice-cold LC-MS grade acetonitrile (Sigma Aldrich) containing 5 microliters of internal standard solution with eight stable isotopic chemicals selected to cover a range of chemical properties. Following addition of acetonitrile, samples were homogenized for a few seconds using an Active Motif EpiShear probe sonicator and equilibrated for 30 min on ice, upon which precipitated proteins were removed by centrifuge (14,000 rpm at 4 degrees C for 10 min). The resulting supernatant (100 microliters) was removed, added to a low volume autosampler vial and maintained at 4 degrees C until analysis (<22 h). |
| Sampleprep Protocol ID: | HRM_SP_082016_01 |
| Sampleprep Protocol Filename: | EmoryUniversity_HRM_sample_preparation_082016_01 |
| Sampleprep Protocol Comments: | Date effective: 30 July 2016 |
| Extraction Method: | 2:1 acetonitrile: sample followed by vortexing and centrifugation |
Combined analysis:
| Analysis ID | AN001776 | AN001777 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | Waters XBridge Amide (50 x 2.1mm,2.5um) | Thermo Higgins C18 (50 x 2.1mm,3um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Fusion Tribrid Orbitrap | Thermo Fusion Tribrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak area | Peak area |
Chromatography:
| Chromatography ID: | CH001255 |
| Chromatography Summary: | The HILIC column is operated parallel to reverse phase column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10-port and 6-port switching valves. During operation of "HILIC separation method, the MS is operated in positive ion mode and 10" microliters of sample is injected onto the HILIC column while the reverse phase column is flushing with wash solution. Flow rate is maintained at 0.35 mL/min "until 1.5 min, increased to 0.4 mL/min at 4 min and held for 1 min. Solvent A is" "100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C" is 2% formic acid (v/v) in LC-MS grade water. Initial mobile phase conditions "are 22.5% A, 75% B, 2.5% C hold for 1.5 min, with linear gradient to 77.5% A," "20% B, 2.5% C at 4 min, hold for 1 min, resulting in a total analytical run time" "of 5 min. During the flushing phase (reverse phase analytical separation), the" "HILIC column is equilibrated with a wash solution of 77.5% A, 20% B, 2.5% C." |
| Methods ID: | 2% formic acid in LC-MS grade water |
| Methods Filename: | 20160920_posHILIC120kres5min_ESI_c18negwash.meth |
| Chromatography Comments: | Triplicate injections for each chromatography mode |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters XBridge Amide (50 x 2.1mm,2.5um) |
| Column Temperature: | 60C |
| Flow Gradient: | A= water, B= acetontrile, C= 2% formic acid in water; 22.5% A, 75% B, 2.5% C hold for 1.5 min, linear gradient to 77.5% A, 20% B, 2.5% C at 4 min, hold for 1 min |
| Flow Rate: | 0.35 mL/min for 1.5 min; linear increase to 0.4 mL/min at 4 min, hold for 1 min |
| Sample Injection: | 10 uL |
| Solvent A: | 100% water |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 5 min |
| Sample Loop Size: | 15 uL |
| Sample Syringe Size: | 100 uL |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH001256 |
| Chromatography Summary: | The C18 column is operated parallel to the HILIC column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10-port and 6- port switching valves. During operation of the "C18 method, the MS is operated in negative ion mode and 10 μL of sample is" injected onto the C18 column while the HILIC column is flushing with wash "solution. Flow rate is maintained at 0.4 mL/min until 1.5 min, increased to 0.5" "mL/min at 2 min and held for 3 min. Solvent A is 100% LC-MS grade water, solvent" B is 100% LC-MS grade acetonitrile and solvent C is 10mM ammonium acetate in "LC-MS grade water. Initial mobile phase conditions are 60% A, 35% B, 5% C hold" "for 0.5 min, with linear gradient to 0% A, 95% B, 5% C at 1.5 min, hold for 3.5" "min, resulting in a total analytical run time of 5 min. During the flushing" "phase (HILIC analytical separation), the C18 column is equilibrated with a wash" "solution of 0% A, 95% B, 5% C until 2.5 min, followed by an equilibration" "solution of 60% A, 35% B, 5% C for 2.5 min." |
| Methods ID: | 10mM ammonium acetate in LC-MS grade water |
| Methods Filename: | 20160920_negC18120kres5min_ESI_HILICposwash.meth |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Thermo Higgins C18 (50 x 2.1mm,3um) |
| Column Temperature: | 60C |
| Flow Rate: | 0.4 mL/min for 1.5 min; linear increase to 0.5 mL/min at 2 min held for 3 min |
| Sample Injection: | 10 uL |
| Solvent A: | 100% water |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 5 min |
| Sample Loop Size: | 15 uL |
| Sample Syringe Size: | 100 uL |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001641 |
| Analysis ID: | AN001776 |
| Instrument Name: | Thermo Fusion Tribrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 250C |
| Collision Gas: | N2 |
| Dry Gas Flow: | 45 |
| Dry Gas Temp: | 150C |
| Mass Accuracy: | < 3ppm |
| Spray Voltage: | 3500 |
| Activation Parameter: | 5.00E+05 |
| Activation Time: | 118ms |
| Interface Voltage: | S-Lens RF level= 55 |
| Resolution Setting: | 120000 |
| Scanning Range: | 85-1275 |
| Analysis Protocol File: | EmoryUniversity_HRM_Fusion-MS_092017_v1.pdf |
| MS ID: | MS001642 |
| Analysis ID: | AN001777 |
| Instrument Name: | Thermo Fusion Tribrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 250C |
| Collision Gas: | N2 |
| Dry Gas Flow: | 45 |
| Dry Gas Temp: | 150C |
| Mass Accuracy: | < 3ppm |
| Spray Voltage: | -4000 |
| Activation Parameter: | 5.00E+05 |
| Activation Time: | 118ms |
| Interface Voltage: | S-Lens RF level= 55 |
| Resolution Setting: | 120000 |
| Scanning Range: | 85-1275 |
| Analysis Protocol File: | EmoryUniversity_HRM_Fusion-MS_092017_v1.pdf |
