Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA211873

Local Sample ID:	8020-S
Subject ID:	SU002300
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	50-90 yrs
Species Group:	Mammals

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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
Species Group:	Mammals

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
8020-S	SA211873	FL025837	Alzheimer patient	Condition
8020-S	SA211873	FL025837	Shikonin	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
Chromatography ID	CH002677	CH002678
MS ID	MS003373	MS003374
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