Metabolic footprinting of tumorigenic and nontumorigenic uroepithelial cells using two-dimensional gas chromatography time-of-flight mass spectrometry

• Published in: Analytical and bioanalytical chemistry Vol. 398; no. 3; pp. 1285 - 1293
• Main Authors: Pasikanti, Kishore Kumar, Norasmara, Juwita, Cai, Shirong, Mahendran, Ratha, Esuvaranathan, Kesavan, Ho, Paul C, Chan, Eric Chun Yong
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.10.2010; Springer-Verlag; Springer
• Subjects: Analysis; Analytical Chemistry; Biochemistry; Biological and medical sciences; Biological markers; Bladder; Bladder cancer; Cancer cells; Cell Line; Cell Proliferation; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatographic methods and physical methods associated with chromatography; Comparative analysis; Culture; Epithelial cells; Exact sciences and technology; Food Science; Fundamental and applied biological sciences. Psychology; Gas chromatographic methods; Gas chromatography; Gas Chromatography-Mass Spectrometry - methods; Genic rearrangement. Recombination. Transposable element; Human; Humans; Laboratory Medicine; Mass spectrometry; Mathematical analysis; Mathematical models; Metabolic footprinting; Metabolites; Metabolomics; Metabonomic analysis; Methods; Molecular and cellular biology; Molecular genetics; Monitoring/Environmental Analysis; Original Paper; Physiological aspects; Spectra; Spectrometric and optical methods; Two dimensional; Two-dimensional gas chromatography time-of-flight mass spectrometry; Urinary Bladder Neoplasms - metabolism; Urinary Bladder Neoplasms - pathology; Urothelium; Urothelium - cytology; Urothelium - metabolism; Singapore; Metabolic profiling; Metabolomics; Metabolic footprinting; Bladder cancer; Two-dimensional gas chromatography time-of-flight mass spectrometry; Metabonomics; Cell culture; Two dimensional chromatography; Metabolite; Supernatant; Artefact; Biology; Identification; Time; Metabolic pathway; Multivariate analysis; Mixture; T-Lymphocyte; Peak; Methanol; Human; Discriminant analysis; Data analysis; Transposable element; In vitro; Protein; Gas chromatography; PLS regression; Phenotype; Sensitivity; Cell line; Time of flight method; Reagents; Technique; Application; Mass spectrometry
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-010-4055-3

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. [graphic removed]