Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA346721

Local Sample ID:	Sen_shHK2_WTHK2_1
Subject ID:	SU003301
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU003301
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
Sen_shHK2_WTHK2_1	SA346721	FL040688	Sen_shHK2_WTHK2	Treatment
Sen_shHK2_WTHK2_1	SA346721	FL040688	lung embryonic fibroblasts	Sample source

Collection:

Collection ID:	CO003294
Collection Summary:	IMR90 primary human diploid lung embryonic fibroblasts were used and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), L-glutamine, sodium pyruvate, non-essential amino acids, sodium bicarbonate and 1% penicillin-streptomycin under low oxygen tension (2%).
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR003310
Treatment Summary:	Stable isotope tracer analysis using 13C6-glucose was conducted in three biologically independent experiments for the following experimental groups: control proliferating cells (Pro), RAS-induced senescent cells via 4-OHT with control shRNA (Sen_shctrl), senescent cells with shRNA targeting HK2 (Sen_shHK2), senescent cells with shRNA targeting HK2 and rescued with wildtype Flag-HK2 (Sen_shHK2_WTHK2), and senescent cells with shRNA targeting HK2 and rescued with mutant Flag-HK2 (Sen_shHK2_MutHK2). The experiments were performed by seeding cells at a density of 3 × 10^5 cells per 6 cm dish and incubating them with 25 mM [13C6]-glucose tracer in glucose-free medium for 30 min.

Treatment ID:

TR003310

Treatment Summary:

Stable isotope tracer analysis using 13C6-glucose was conducted in three biologically independent experiments for the following experimental groups: control proliferating cells (Pro), RAS-induced senescent cells via 4-OHT with control shRNA (Sen_shctrl), senescent cells with shRNA targeting HK2 (Sen_shHK2), senescent cells with shRNA targeting HK2 and rescued with wildtype Flag-HK2 (Sen_shHK2_WTHK2), and senescent cells with shRNA targeting HK2 and rescued with mutant Flag-HK2 (Sen_shHK2_MutHK2). The experiments were performed by seeding cells at a density of 3 × 10^5 cells per 6 cm dish and incubating them with 25 mM [13C6]-glucose tracer in glucose-free medium for 30 min.

Sample Preparation:

Sampleprep ID:	SP003308
Sampleprep Summary:	Following incubation, the cells were washed with chilled PBS and incubated with 500 µl of extraction solution (80:20 v/v methanol/water) at 4 °C for 5 minutes. Next, cells were scraped with a polypropylene cell scraper and the extraction solution from each sample was collected, vortexed, and incubated on dry ice for at least 30 min. Then, each sample was centrifuged at maximum speed at 4 °C for 10 minutes, and the resulting supernatant was used for metabolic measurements.

Sampleprep ID:

SP003308

Sampleprep Summary:

Following incubation, the cells were washed with chilled PBS and incubated with 500 µl of extraction solution (80:20 v/v methanol/water) at 4 °C for 5 minutes. Next, cells were scraped with a polypropylene cell scraper and the extraction solution from each sample was collected, vortexed, and incubated on dry ice for at least 30 min. Then, each sample was centrifuged at maximum speed at 4 °C for 10 minutes, and the resulting supernatant was used for metabolic measurements.

Combined analysis:

Analysis ID	AN005226
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish Horizon UHPLC
Column	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF-X Orbitrap
Ion Mode	UNSPECIFIED
Units	Peak Area

Chromatography:

Chromatography ID:	CH003953
Chromatography Summary:	Hydrophilic interaction liquid chromatography (HILIC) was performed at 0.2 ml/min on a ZIC-pHILIC column (2.1 mm × 150 mm, 5 µm particle size, EMD Millipore) at 45 °C. Solvent A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide, pH 9.2, and solvent B was acetonitrile. The gradient was 85% B for 2 min, 85% B to 20% B over 15 min, 20% B to 85% B over 0.1 min, and 85% B for 8.9 min. The autosampler was held at 4 °C. For each analysis, 4 µl of sample was injected.
Instrument Name:	Thermo Vanquish Horizon UHPLC
Column Name:	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:	45
Flow Gradient:	85% B for 2 min, 85% B to 20% B over 15 min, 20% B to 85% B over 0.1 min, and 85% B for 8.9 min
Flow Rate:	0.2 ml/min
Solvent A:	100% water; 20 mM ammonium carbonate; 5 µM medronic acid; 0.1% ammonium hydroxide
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS004959
Analysis ID:	AN005226
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The following parameters were used for the MS analysis: sheath gas flow rate, 40; auxiliary gas flow rate, 10; sweep gas flow rate, 2; auxiliary gas heater temperature, 350 °C; spray voltage, 3.5 kV for positive mode and 3.2 kV for negative mode; capillary temperature, 325 °C; and funnel RF level, 40. All samples were analyzed by full MS with polarity switching. Technical injections of an unlabeled sample pool were analyzed throughout the sample sequence. The unlabeled sample pool was also analyzed by data-dependent MS/MS with separate runs for positive and negative ion modes. Full MS scans were acquired at 120,000 resolution with a scan range of 65-975 m/z. Data-dependent MS/MS scans were acquired for the top 10 highest intensity ions at 15,000 resolution with an isolation width of 1.0 m/z and stepped normalized collision energy of 20-40-60. Annotation and quantitation of metabolites and carbon isotopologues with natural isotope abundance correction were performed using Compound Discoverer 3.3 software (Thermo Scientific). Metabolite measurements were normalized based on the protein concentration in the protein pellets.
Ion Mode:	UNSPECIFIED