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MB Sample ID: SA328110

Local Sample ID:2_6
Subject ID:SU003144
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

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Subject:

Subject ID:SU003144
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
2_6SA328110FL038950Young-wildtypeGenotype
2_6SA328110FL038950Sham young+MIF20Treatment

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
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