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MB Sample ID: SA211883

Local Sample ID:8011-OE
Subject ID:SU002300
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:50-90 yrs

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Subject:

Subject ID:SU002300
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:50-90 yrs

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
8011-OESA211883FL025840non-demented controlCondition
8011-OESA211883FL025840PKM2-overexpressionTreatment

Collection:

Collection ID:CO002293
Collection Summary:For Metabolome, iNs were flow cytometry-isolated 21 days of conversion. iNs were sorted based on the expression of the PSA-NCAM surface markers using PSA-NCAM-PE (Miltenyi) and re-plated on Geltrex-coated wells. The following day, iNs were treated with 2.5 mM DGlucose-C13 for 6 hours and harvested using Tryple. Supernatant and cell pellets were shock frozen in liquid nitrogen.
Sample Type:Biopsy

Treatment:

Treatment ID:TR002312
Treatment Summary:After two weeks of conversion, cells were treated for 10 days with shikonin (10 ┬ÁM, ChemCruz) before FACS sorting. Control iNs were FACS-sorted and plated on Geltrex-coated wells before transduction with pLVXTP-EGFP-PKM2 or pLVXTP-EGFP. Around 60 % of all neurons were successfully transduced. Five days after transduction, iNs were harvested for MS-analysis. Alternatively, cells were treated with CoDo for 6 hours before harvest.

Sample Preparation:

Sampleprep ID:SP002306
Sampleprep Summary:Metabolites from frozen cell pellets were extracted at 150,000 cells/mL in cold 5:3:2 MeOH:acetonitrile:water. Samples were vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C). Supernatants were split into two equal tubes and gently dried using a vacuum concentrator. One set of residues were resuspended in 12 uL of 0.1% formic acid (for positive ion mode). The other set of residues were resuspended in 12 uL of 1 mM ammonium acetate (for negative ion mode).

Combined analysis:

Analysis ID AN003622 AN003623
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
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:CH002677
Chromatography Summary:Negative C18 cells
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Sample Injection:10 uL
Chromatography Type:Reversed phase
  
Chromatography ID:CH002678
Chromatography Summary:Positive C18 cells
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Sample Injection:10 uL
Chromatography Type:Reversed phase

MS:

MS ID:MS003373
Analysis ID:AN003622
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:NEGATIVE
  
MS ID:MS003374
Analysis ID:AN003623
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:POSITIVE
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