Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA333654

Local Sample ID:	SUB13607p4_SPL08
Subject ID:	SU003221
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU003221
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
SUB13607p4_SPL08	SA333654	FL039616	DMSO	Treatment
SUB13607p4_SPL08	SA333654	FL039616	ICC13-7 cells	Sample source

Collection:

Collection ID:	CO003214
Collection Summary:	media was completely aspirated, and cells were washed with ice-cold saline quickly. After washing, fully remove saline and cells can be scrapped in 1 ml pre-cooled methanol (-20°C) with internal standards (Cambridge Isotope Laboratories, MSK-A2-1.2), and transferred to glass vials, stored at -80°C until extraction.
Sample Type:	ICC13-7

Treatment:

Treatment ID:	TR003230
Treatment Summary:	Cells were pretreated with DMSO or 100nM Infigratinib for 24h in lipid-depleted media. Then they were changed to 13C-Plamitate labeling media for 8h (with DMSO or infigratinib) before harvesting cells.

Sample Preparation:

Sampleprep ID:	SP003227
Sampleprep Summary:	extractions were conducted following the biphasic extraction protocol. Basically, add cold chloroform to samples (in methanol) with a ratio of 2:1. Vortex samples for 1 minute to homogenize. Add 1 fraction of water, and vortex samples again. Glass vials were centrifuged at 3000 rcf for 10 minutes for phase separation. The aqueous phase was transferred to a new glass vial for metabolomics and the remaining interphase was used for protein quantification. The aqueous phase was evaporated under nitrogen flow. Samples were resuspended in 50% acetonitrile, and the volume was scaled according to the protein amounts. 15ul was used for the lowest biomass, and all others were scaled accordingly. Standard mixes were prepared at 100 uM and run after the samples to allow for the identification of the targets.

Sampleprep ID:

SP003227

Sampleprep Summary:

extractions were conducted following the biphasic extraction protocol. Basically, add cold chloroform to samples (in methanol) with a ratio of 2:1. Vortex samples for 1 minute to homogenize. Add 1 fraction of water, and vortex samples again. Glass vials were centrifuged at 3000 rcf for 10 minutes for phase separation. The aqueous phase was transferred to a new glass vial for metabolomics and the remaining interphase was used for protein quantification. The aqueous phase was evaporated under nitrogen flow. Samples were resuspended in 50% acetonitrile, and the volume was scaled according to the protein amounts. 15ul was used for the lowest biomass, and all others were scaled accordingly. Standard mixes were prepared at 100 uM and run after the samples to allow for the identification of the targets.

Combined analysis:

Analysis ID	AN005085
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Orbitrap ID-X tribrid
Ion Mode	UNSPECIFIED
Units	area analyzed by Compound discoverer (counts x seconds)

Chromatography:

Chromatography ID:	CH003841
Instrument Name:	Thermo Vanquish
Column Name:	Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
Column Temperature:	40
Flow Gradient:	Started at 93% B and 7% A ; 40% B/60% A in 19 min; 100% A in 9 min; 100% A for 5 min; back to 93% B/7% A in 3 min; re-equilibration at 93% B/7% A for 9 min.
Flow Rate:	0.05 to 0.15 mL/min in 30 sec; then held at 0.15mL/min
Solvent A:	100% Water; 20 mMAmmonium Carbonate, 0.1% Ammonium hydroxide
Solvent B:	Acetonitrile 97%, 3% water
Chromatography Type:	HILIC

MS:

MS ID:	MS004822
Analysis ID:	AN005085
Instrument Name:	Thermo Orbitrap ID-X tribrid
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data was acquired on the ID-X in switching polarities at 120000 resolution, with an AGC target of 1e5, and a m/z range of 65 to 1000. MS1 data is acquired in switching polarities for all samples.Data is analyzed in Compound Discoverer 3.3 (CD, ThermoFisher Scientific). A mix of standard of each target was run along the samples and used as the unlabeled reference for the CD labelling workflow. Isotopomer distribution is found in the exchange column, as % of 0 labelled carbon, 1 labelled carbon, 2 labelled carbon, etc. Those are corrected for natural abundance. For the compounds that couldn’t be analyzed by CD, the areas have been extracted with Tracefinder manually.
Ion Mode:	UNSPECIFIED