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MB Sample ID: SA163491
Local Sample ID: | 508 |
Subject ID: | SU001824 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
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Subject:
Subject ID: | SU001824 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
508 | SA163491 | FL019462 | sham | group |
508 | SA163491 | FL019462 | 7d | time point |
Collection:
Collection ID: | CO001817 |
Collection Summary: | Adult (8-10 week-old), male C57B/6 mice (Jackson Laboratories, Bar Harbor, ME), weighing between 20-25g were used. They were maintained on a standard diet and water was freely available. All experiments were conducted in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal protocol was approved by the Animal Care and Use Committee of the University of Colorado, Denver. Surgical Protocol. To induce ischemic AKI (29), mice were anesthetized with intraperitoneal avertin (2,2,2-tribromoethanol; Sigma Aldrich, Milwaukee, WI), a laparotomy was performed, and both renal pedicles were clamped for 22 minutes. Mice received 500 µl saline with buprenex subcutaneous injection preceding surgery and 500 µl saline was administered by subcutaneous injection daily after surgery. The sham procedure is similar in all respects – including laparotomy - except that renal pedicle clamping is not performed. Collection and preparation of plasma and lung samples. Blood was obtained via cardiac puncture and centrifuged at 3000g at 4°C for ten minutes; plasma was collected and centrifuged a second time at 3000g for one minute. The lungs were collected, weighed, snap frozen in liquid nitrogen and stored at -80°C. In this experiment, lung, heart, kidney and liver were all rapidly collected and snap frozen for future metabolomics assessment (19); in order to limit time to freezing (and potential changes in metabolic phenotype due to death) no additional processing of tissue occurred and organs were not perfused prior to collection. |
Sample Type: | Lung |
Treatment:
Treatment ID: | TR001837 |
Treatment Summary: | To induce ischemic AKI (29), mice were anesthetized with intraperitoneal avertin (2,2,2-tribromoethanol; Sigma Aldrich, Milwaukee, WI), a laparotomy was performed, and both renal pedicles were clamped for 22 minutes. Mice received 500 µl saline with buprenex subcutaneous injection preceding surgery and 500 µl saline was administered by subcutaneous injection daily after surgery. The sham procedure is similar in all respects – including laparotomy - except that renal pedicle clamping is not performed. |
Sample Preparation:
Sampleprep ID: | SP001830 |
Sampleprep Summary: | Metabolomics analyses. Frozen lung tissue was milled with mortar and pestle in the presence of liquid nitrogen and weighed to the nearest 0.1 mg. At a tissue concentration of 40 mg/mL, the samples were extracted in ice-cold lysis/extraction buffer (5:3:2 MeOH:MeCN:water v/v/v) followed by agitation at 4°C for 30 minutes and centrifugation at 18,213 g for 10 minutes at 4°C. The supernatants (10 uL per injection) were immediately analyzed by UHPLC-MS. |
Combined analysis:
Analysis ID | AN002843 | AN002844 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
Column | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
Ion Mode | NEGATIVE | POSITIVE |
Units | peak area | peak area |
Chromatography:
Chromatography ID: | CH002104 |
Chromatography Summary: | 10 μl of tissue extracts were injected into a UHPLC system (Ultimate 3000, Thermo, San Jose, CA, USA) and separated through a 3 min isocratic elution on a Kinetex XB-C18 column (150 × 2.1 mm i.d., 1.7 μm particle size – Phenomenex, Torrance, CA, USA) at 250 μl/min (mobile phase: 5% acetonitrile, 95% 18 mΩ H2O, 0.1% formic acid; column temperature: 25°C). |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
Column Temperature: | 25 |
Flow Gradient: | isocratic |
Flow Rate: | 251 ul/min |
Solvent A: | 5% acetonitrile/95%water; 0.1% formic acid |
Solvent B: | isocratic |
Chromatography Type: | Reversed phase |
Chromatography ID: | CH002105 |
Chromatography Summary: | 10 μl of tissue extracts were injected into a UHPLC system (Ultimate 3000, Thermo, San Jose, CA, USA) and separated through a 3 min isocratic elution on a Kinetex XB-C18 column (150 × 2.1 mm i.d., 1.7 μm particle size – Phenomenex, Torrance, CA, USA) at 250 μl/min (mobile phase: 5% acetonitrile, 95% 18 mΩ H2O, 0.1% formic acid; column temperature: 25°C). |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
Column Temperature: | 25 |
Flow Gradient: | isocratic |
Flow Rate: | 252 ul/min |
Solvent A: | 5% acetonitrile/95%water; 0.1% formic acid |
Solvent B: | isocratic |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS002636 |
Analysis ID: | AN002843 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs. MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above. |
Ion Mode: | NEGATIVE |
MS ID: | MS002637 |
Analysis ID: | AN002844 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs. MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above. |
Ion Mode: | POSITIVE |