Summary of Study ST002703

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001674. The data can be accessed directly via it's Project DOI: 10.21228/M8B14V This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002703
Study Title	Multi-Omics Analysis Revealed a Significant Molecular Changes in Doxorubicin-Resistant Lung Cancer Cells.
Study Type	LC/MS/MS
Study Summary	Lung cancer is the second most common cancer and the leading cause of cancer-related deaths worldwide. Chemotherapy resistance in lung cancer is one of the major characteristics of an aggressive phenotype with poor prognosis. Therefore, there is a critical need to explore the significant molecular changes associated with resistance to conventional chemotherapy, and identify potential therapeutic targets for treatment of resistant lung cancer. In this study, we have performed comprehensive quantitative proteomics and metabolomics analysis of non-small cell lung cancer cells (A549-P) and doxorubicin resistant A549 cells (A549-R), using state-of-the-art Trapped Ion Mobility Spectroscopy, Time-of-Flight Mass Spectrometry (TIMS-TOF-MS). The results revealed 30 dysregulated proteins and 37 significantly altered metabolites in A549-R cells compared to A549-P cells. Among the significantly upregulated proteins are liver carboxylesterase 1, anterior gradient protein 2 homolog and nicotinamide phosphoribosyltransferase. A group of the upregulated proteins are endogenous and xenobiotic-metabolizing enzymes, including UDP-glucuronosyltransferase 1-6, CES1, and epoxide hydrolase 1. While Importin, ATP-citrate synthase and CTP synthase are downregulated. The significantly altered metabolites include sepiapterin, glutathione, glycine, pyridine and niacinamide. The performed multi-omics integrated analysis revealed the involvement of purine and glutathione metabolism, ABC transporters, citric acid cycle in the development of resistance in A549 cells, besides the involvement of energy metabolism, pathways related to cancer progression, invasion and migration, and redox homeostasis. Collectively, this exploratory study effectively revealed the significantly dysregulated proteins and metabolites in doxorubicin resistant A549 cells and shed the light on potential biomarkers for chemotherapy resistant non-small cell lung cancer. In addition, multi-omics integrated analysis elucidates the involved pathways in resistance including pathways related to progression and invasion which would improve prognosis and open the door for new potential therapeutic targets.
Institute	University of Sharjah
Department	Research institute of medical and health science
Laboratory	Biomarker Discovery Group
Last Name	Facility
First Name	Core
Address	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, UAE, Sharjah, 000, United Arab Emirates
Email	tims-tof@sharjah.ac.ae
Phone	+971 6 5057656
Submit Date	2023-05-04
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2023-11-30
Release Version	1

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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.