Metabolic footprinting of tumorigenic and nontumorigenic uroepithelial cells using two-dimensional gas chromatography time-of-flight mass spectrometry |
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Authors: | Kishore Kumar Pasikanti Juwita Norasmara Shirong Cai Ratha Mahendran Kesavan Esuvaranathan Paul C Ho Eric Chun Yong Chan |
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Institution: | (1) Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore, 117543, Republic of Singapore;(2) Department of Surgery, National University Hospital, Singapore, 119074, Republic of Singapore;(3) Department of Urology, National University Hospital, Singapore, 119074, Republic of Singapore; |
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Abstract: | In this study, gas chromatography mass spectrometry (GC-MS) and two-dimensional gas chromatography time-of-flight mass spectrometry
(GC×GC-TOFMS) were employed for the metabolic footprinting of a pair of immortalized human uroepithelial cells namely HUC-1
(nontumorigenic) and HUC T-2 (tumorigenic). Both HUC-1 and HUC T-2 cell lines were cultivated in 1 mL of Ham’s F-12 media.
Subsequent to 48 h of incubation, 200 μL of cell culture supernatant was protein-precipitated using 1.7 mL of methanol and
an aliquot of 1.5 mL of the mixture was separated, dried, trimethylsilyl-derivatized, and analyzed using GC-MS and GC×GC-TOFMS.
Metabolic profiles were analyzed using multivariate data analysis techniques to evaluate the changes of the metabolomes. Both
GC-MS and GC×GC-TOFMS analyses showed distinct differences in metabolic phenotypes of the normal and tumorigenic human bladder
cells (partial least squares-discriminant analysis (PLS-DA) of GC×GC-TOFMS data; two latent variables, R
2
X = 0.418, R
2
Y = 0.977 and Q
2 (cumulative) = 0.852). Twenty metabolites were identified as being statistically different between the two cell types. These
metabolites revealed that several key metabolic pathways were perturbed in tumorigenic urothelial cells as compared to the
normal cells. Application of GC×GC-TOFMS offered several advantages compared to classical one-dimensional GC-MS which include
enhanced chromatographic resolution (without increase in analytical run time), increase in sensitivity, improved identification
of metabolites, and also separation of reagent artifacts from the metabolite peaks. Our results reinforced the advantages
of GC×GC-TOFMS and the role of metabolomics in characterizing bladder cancer biology using in vitro cell culture models. |
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Keywords: | |
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