Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA271294

Local Sample ID:	R Control 1A-01_13_1_3229
Subject ID:	SU002808
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002808
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
R Control 1A-01_13_1_3229	SA271294	FL035202	A549-P	Treatment

Collection:

Collection ID:	CO002801
Collection Summary:	The human non-small cell lung cancer cell line (A549) utilized in this study was purchased from AmericanType Culture Collection (ATCC) (Manassas, USA). A doxorubicin-resistant A549 cell line was generated in our laboratory (Drug Design and Discovery Research Group, Pharmacology Laboratory, Sharjah Institute for Medical Research, University of Sharjah). Doxorubicin (Dox) and irinotecan were purchased from Sigma-Aldrich (Darmstadt, Germany) and Abcam (Cambridge, UK), respectively. A Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (1:1) (DMEM/F-12) medium was purchased from Gibco, Thermo Fisher Scientific (Massachusetts, USA). Fetal bovine serum (FBS), penicillin/streptomycin, trypsin, and trypan blue were purchased from Sigma-Aldrich (Darmstadt, Germany). For cell viability assay: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Darmstadt, Germany). Protease inhibitor cocktail tablets were purchased from Roche (Mannheim, Germany). Methanol (≥99.9%), acetonitrile (ACN) and deionized water, as well as LC-MS CHROMASOLV, were purchased from Honeywell (Seelze, Germany). Trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich (Darmstadt, Germany) and acetonitrile (ACN) was purchased from Honeywell (Charlotte, USA). Lys-C endopeptidase was purchased from Fujifilm Wako Chemicals (Virginia, USA). For western blot analysis: primary antibodies against liver carboxylesterase 1/CES1 and β-actin were obtained from Abcam (Cambridge, UK) and Cell Signaling Technologies (Danvers, USA), respectively. Anti-rabbit IgG, Horseradish Peroxidase (HRP)-linked secondary antibody was purchased from Cell Signaling Technologies (Danvers, USA). Skimmed milk was purchased from Sigma-Aldrich (Darmstadt, Germany). For Enzyme-linked immunosorbent assay (ELISA): carboxylesterase 1 (CES1) specific activity assay kit purchased from Abcam (Cambridge, UK).
Sample Type:	Lung

Treatment:

Treatment ID:	TR002817
Treatment Summary:	The human non-small cell lung cancer cell line (A549) was purchased from AmericanType Culture Collection (ATCC) (Manassas, USA). A doxorubicin-resistant A549 (A549-R) cell line was generated in our laboratory (Drug Design and Discovery Research Group, Pharmacology Laboratory, Sharjah Institute for Medical Research, University of Sharjah). To develop resistant cells from the parental A549 cells (A549-P), A549-P cells were treated with a tenth of Dox's maximal inhibitory concentration (IC10). The surviving population of cells was cultured in a new flask and treated with a gradual increase of Dox concentration for 8 months. Maintenance of A549-R cells resistance was at a dose of 0.02 µg/ml of Dox. A549-P and A549-R cells were cultured in a Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (1:1) medium (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were incubated in a humidified atmosphere of 5% CO2 at 37 ˚C.

Treatment ID:

TR002817

Treatment Summary:

The human non-small cell lung cancer cell line (A549) was purchased from AmericanType Culture Collection (ATCC) (Manassas, USA). A doxorubicin-resistant A549 (A549-R) cell line was generated in our laboratory (Drug Design and Discovery Research Group, Pharmacology Laboratory, Sharjah Institute for Medical Research, University of Sharjah). To develop resistant cells from the parental A549 cells (A549-P), A549-P cells were treated with a tenth of Dox's maximal inhibitory concentration (IC10). The surviving population of cells was cultured in a new flask and treated with a gradual increase of Dox concentration for 8 months. Maintenance of A549-R cells resistance was at a dose of 0.02 µg/ml of Dox. A549-P and A549-R cells were cultured in a Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (1:1) medium (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were incubated in a humidified atmosphere of 5% CO2 at 37 ˚C.

Sample Preparation:

Sampleprep ID:	SP002814
Sampleprep Summary:	Two million cells from both cell lines (A549-P and A549-R) were seeded in T75 cell culture flasks. Following culturing the cells, they were collected by trypsin and a pellet of 3x106 cells was stored at -80 ℃ for further analysis. Each cell line was prepared in triplicates for the metabolomics and proteomics studies. Frozen A549-P and A549-R cells pellets were centrifuged at 14000 rpm for 5 min at 4 ℃ to separate them from Phosphate Buffered Saline (PBS) buffer. The pellets were suspended in a lysis buffer (10 mM Tris) and protease inhibitor cocktail tablets. The lysed cells were kept on ice for 10 min, and then each sample was vortexed for 30 s. After that, the samples were sonicated in an ice bath using a Q500 sonicator from QSonica Sonicator (Fairfield, Connecticut, USA) at 30% amp for 30 s. Then, the samples were centrifuged at 14000 rpm for 5 min at 4℃, 400 µL of methanol was added to the supernatant, followed by 300 µL of chloroform and vortexed for 30 seconds, and then centrifuged at 14000 rpm for 5 min at 4 ℃. The resulting aqueous biphasic solution's upper layer was transferred carefully into LC vials without touching the white disk for metabolites analysis. For proteins extraction, the generated white disk and the lower layer were washed with 300 µL of methanol and vortexed vigorously, and then centrifuged at 14000 rpm for 3 min at 4 ℃. After that, the protein pellet was precipitated and allowed to dry by keeping the tubes opened for 5 min. Then a denaturation buffer was used to re-suspend the pellets (10 mM Tris, 6 M Urea, 2 M Thiourea, pH 8) and the concentration of precipitated proteins was determined using the modified Bradford assay. The resulted supernatant from the previous centrifugation, which contains metabolites, was transferred to the previously collected metabolites in the LC vials. The metabolites samples were dried at 45 ℃ using an EZ-2 Plus evaporator from GeneVac (Ipswich, UK), and then the dehydrated extracts were reconstituted in 200 µL 0.1% formic acid (FA) in water and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic syringe filter with a pore size of 0.45 µm and were ready to be analyzed using Q-TOF MS.

Sampleprep ID:

SP002814

Sampleprep Summary:

Two million cells from both cell lines (A549-P and A549-R) were seeded in T75 cell culture flasks. Following culturing the cells, they were collected by trypsin and a pellet of 3x106 cells was stored at -80 ℃ for further analysis. Each cell line was prepared in triplicates for the metabolomics and proteomics studies. Frozen A549-P and A549-R cells pellets were centrifuged at 14000 rpm for 5 min at 4 ℃ to separate them from Phosphate Buffered Saline (PBS) buffer. The pellets were suspended in a lysis buffer (10 mM Tris) and protease inhibitor cocktail tablets. The lysed cells were kept on ice for 10 min, and then each sample was vortexed for 30 s. After that, the samples were sonicated in an ice bath using a Q500 sonicator from QSonica Sonicator (Fairfield, Connecticut, USA) at 30% amp for 30 s. Then, the samples were centrifuged at 14000 rpm for 5 min at 4℃, 400 µL of methanol was added to the supernatant, followed by 300 µL of chloroform and vortexed for 30 seconds, and then centrifuged at 14000 rpm for 5 min at 4 ℃. The resulting aqueous biphasic solution's upper layer was transferred carefully into LC vials without touching the white disk for metabolites analysis. For proteins extraction, the generated white disk and the lower layer were washed with 300 µL of methanol and vortexed vigorously, and then centrifuged at 14000 rpm for 3 min at 4 ℃. After that, the protein pellet was precipitated and allowed to dry by keeping the tubes opened for 5 min. Then a denaturation buffer was used to re-suspend the pellets (10 mM Tris, 6 M Urea, 2 M Thiourea, pH 8) and the concentration of precipitated proteins was determined using the modified Bradford assay. The resulted supernatant from the previous centrifugation, which contains metabolites, was transferred to the previously collected metabolites in the LC vials. The metabolites samples were dried at 45 ℃ using an EZ-2 Plus evaporator from GeneVac (Ipswich, UK), and then the dehydrated extracts were reconstituted in 200 µL 0.1% formic acid (FA) in water and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic syringe filter with a pore size of 0.45 µm and were ready to be analyzed using Q-TOF MS.

Combined analysis:

Analysis ID	AN004383
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker timsTOF
Column	Hamilton Intensity Solo 2 C18(100 x 2.1 mm, 1.8 um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH003288
Chromatography Summary:	Then, liquid chromatography separation was done using Intensity Solo 1.8 C18-2 column (100×2.1 mm, 1.8 μm) from Bruker Daltonik GmbH (Bremen, Germany). The column temperature was kept at 35 ℃ and the flow rate was constant at 0.3 ml/min. The mobile phase composed of 0.1% FA in HPLC grade water (solvent A) and 0.1% FA in ACN (solvent B).
Instrument Name:	Bruker timsTOF
Column Name:	Hamilton Intensity Solo 2 C18(100 x 2.1 mm, 1.8 um)
Column Temperature:	35
Flow Gradient:	1% B was held for 2 min, ramping to 99% B over 15 min, and held at 99% B for 3 min before re-equilibrating to 1% B for 10 min
Flow Rate:	250 uL/min
Solvent A:	Water (0.1% Formic Acid)
Solvent B:	ACN (0.1% Formic Acid)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004132
Analysis ID:	AN004383
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	MetaboScape® 4.0 software from Bruker (Bremen, Germany) was used for data processing. The parameters for the detection were as follows: a minimum of 7 spectra were used for peak detection, while for molecular features detection, the minimum intensity threshold was equivalent to 1000 counts. Mass recalibration was performed within a retention time range between 0-0.3 min. Only those features present in at least 2 of 6 samples were considered. Finally, the detected MS/MS spectra assigned onto the bucket table allowed for better viewing and understanding. The main features included retention time, measured m/z, detected fragments, and molecular weight [12]. Data bucketing parameters were as follows: the retention time range was between 0.3 min and 25 min, whereas the mass range began at 50 m/z and finished at 1,300 m/z. A two-tailed independent student’s t-test was utilized to determine the significantly altered metabolites between the two cell lines. The threshold for significance was p-value <0.05. Functional enrichment analysis was constructed utilizing the MetaboAnalyst 5.0 website (https://www.metaboanalyst.ca).
Ion Mode:	POSITIVE