Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA211876

Local Sample ID:	8011-DFO
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 ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
8011-DFO	SA211876	FL025838	non-demented control	Condition
8011-DFO	SA211876	FL025838	CoDo Treatment	Treatment

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 µM, 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.

Treatment ID:

TR002312

Treatment Summary:

After two weeks of conversion, cells were treated for 10 days with shikonin (10 µM, 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).

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