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MB Sample ID: SA328106
Local Sample ID: | 1_1 |
Subject ID: | SU003144 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003144 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
1_1 | SA328106 | FL038949 | Young-wildtype | Genotype |
1_1 | SA328106 | FL038949 | Sham young | Treatment |
Collection:
Collection ID: | CO003137 |
Collection Summary: | The sample collection method was as follows: All mouse groups underwent anesthesia with 2%-3% isoflurane and 100% O2. The mouse hearts were excised and rinsed in ice-cold PBS. Both atriums were removed; only the left ventricular myocardium, including the infarct area, was collected and was quickly frozen by using liquid nitrogen. Samples were preserved below -80 °C and were sent out for metabolomic analysis. |
Sample Type: | Heart |
Treatment:
Treatment ID: | TR003153 |
Treatment Summary: | Sham young;Sham young+MIF20;Young I/R;Young I/R+MIF20;Sham aged;Sham aged+MIF20;Aged I/R;Aged I/R +MIF20 |
Sample Preparation:
Sampleprep ID: | SP003150 |
Sampleprep Summary: | The metabolomic sample preparation method was: The tissue samples were ground into a homogenous powder with a Retsch Cryomill (Retsch-Allee, Haan, Germany). 45 – 80 mg of the pulverized sample was weighed and stored in the -80 oC freezer prior to extraction. Samples were allowed to thaw at 4 °C for 30 min before adding 1.5 mL of 20:40:40 water/methanol/acetonitrile with 0.1 M formic acid for metabolites extraction. The samples were vortexed and centrifuged and the supernatants were isolated and dried completely under nitrogen. The dried samples were resuspended in 300 µL water and an aliquot were transferred to autosampler vials for UHPLC-HRMS analysis. Mass analysis was carried out at the University of Tennessee Biological and Small Molecule Mass Spectrometry Core (RRID: SCR_021368) using an UltiMate 3000 liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled to an Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). |
Combined analysis:
Analysis ID | AN004967 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Thermo Dionex Ultimate 3000 |
Column | Phenomenex Synergi Hydro-RP (100 x 2mm,2.5um) |
MS Type | ESI |
MS instrument type | Orbitrap |
MS instrument name | Thermo Exactive Plus Orbitrap |
Ion Mode | NEGATIVE |
Units | Peak area |
Chromatography:
Chromatography ID: | CH003749 |
Chromatography Summary: | Mass analysis was carried out at the University of Tennessee Biological and Small Molecule Mass Spectrometry Core (RRID: SCR_021368) using an UltiMate 3000 liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled to an Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Metabolites were separated using a Synergi Hydro RP column (2.5 μm, 100 × 2.0 mm; Phenomenex, Torrance, CA, USA) using a reversed phase ion pairing chromatographic method. This method uses a tributylamine ion pairing reagent with a water: methanol solvent gradient elution within a 25-minute duration as reported previously (Bazurto et al, 2018: Bazurto JV, Dearth SP, Tague ED, Campagna SR, Downs DM. Untargeted metabolomics confirms and extends the understanding of the impact of aminoimidazole carboxamide ribotide (AICAR) in the metabolic network of Salmonella enterica. Microb Cell. 2017 Nov 22;5(2):74-87. doi: 10.15698/mic2018.02.613. PMID: 29417056; PMCID: PMC5798407.). |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Phenomenex Synergi Hydro-RP (100 x 2mm,2.5um) |
Column Temperature: | 25 |
Flow Gradient: | 0 to 5 min 0% B, from 5 to 13 min 20% B, from 13 to 15.5 min 55% B, from 15.5 to 19 min 95% B, and from 19 to 25 min 0% B |
Flow Rate: | 200 µL/min |
Solvent A: | 97:3 LCMS grade water : methanol, 11 mM tributylamine, 15 mM acetic acid. |
Solvent B: | LCMS grade methanol |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS004707 |
Analysis ID: | AN004967 |
Instrument Name: | Thermo Exactive Plus Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Mass analysis was carried out at the University of Tennessee Biological and Small Molecule Mass Spectrometry Core (RRID: SCR_021368) using an UltiMate 3000 liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled to an Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Metabolites were separated using a Synergi Hydro RP column (2.5 μm, 100 × 2.0 mm; Phenomenex, Torrance, CA, USA) using a reversed phase ion pairing chromatographic method. This method uses a tributylamine ion pairing reagent with a water: methanol solvent gradient elution within a 25-minute duration as reported previously (Bazurto et al., 2018). All solvents used were LC-MS grade. The chromatographic gradient was from 0 to 5 min 0% B, from 5 to 13 min 20% B, from 13 to 15.5 min 55% B, from 15.5 to 19 min 95% B, and from 19 to 25 min 0% B. (ref to paper: (Bazurto et al, 2018)) The separated metabolites were ionized using electrospray ionization with negative polarity and the full scan mass analysis was performed with a resolution of 140,000 as previously reported. The Xcalibur (RAW) files generated from the UPLC-HRMS analysis were converted to the mzML format using the msconvert software to enable data centroiding. Metabolomic Analysis and Visualization Engine (MAVEN) was used to integrate the peak areas from the extracted ion chromatograms (EIC). Prior to identification and integration, MAVEN was used to perform a nonlinear retention time correction across all samples. Metabolites were identified by comparing chromatographic retention time and exact masses within ± 5 ppm mass tolerance to an in-house standard library. Identifications were validated using the natural abundance of isotopes in the compound. The integrated peak areas were used for further statistical and biological analysis. Unidentified spectral features were annotated and analyzed using online LC-MS raw data spectra processing features in MetaboAnalyst 5.0. |
Ion Mode: | NEGATIVE |