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MB Sample ID: SA163464

Local Sample ID:553
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 IDMB Sample IDFactor Level IDLevel ValueFactor Name
553SA163464FL019459normalgroup
553SA163464FL01945924htime 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
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