Analytical and Bioanalytical Chemistry

, Volume 398, Issue 3, pp 1285–1293 | Cite as

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

  • Kishore Kumar Pasikanti
  • Juwita Norasmara
  • Shirong Cai
  • Ratha Mahendran
  • Kesavan Esuvaranathan
  • Paul C. Ho
  • Eric Chun Yong ChanEmail author
Original Paper


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.


Metabolic footprinting of tumorigenic and nontumorigenic uroepithelial cells using GCxGCTOFMS


Metabolomics Metabolic footprinting Metabolic profiling Two-dimensional gas chromatography time-of-flight mass spectrometry Bladder cancer Metabonomics 


Acknowledgments of research support

This study was supported by the National University of Singapore (NUS) grant R-148-000-100-112 provided to E.C.Y.C and National Medical Research Council grant R-176-000-119-213 provided to K.E., P.C.H., R.M., and E.C.Y.C. GC × GC-TOFMS was kindly sponsored by the NUS grant R-279-000-249-646. K.K.P is supported by NUS President's Graduate Fellowship.

Supplementary material

216_2010_4055_MOESM1_ESM.pdf (675 kb)
ESM 1  (PDF 675 kb)


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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Kishore Kumar Pasikanti
    • 1
  • Juwita Norasmara
    • 2
  • Shirong Cai
    • 2
  • Ratha Mahendran
    • 2
  • Kesavan Esuvaranathan
    • 2
    • 3
  • Paul C. Ho
    • 1
  • Eric Chun Yong Chan
    • 1
  1. 1.Department of Pharmacy, Faculty of ScienceNational University of SingaporeSingaporeRepublic of Singapore
  2. 2.Department of SurgeryNational University HospitalSingaporeRepublic of Singapore
  3. 3.Department of UrologyNational University HospitalSingaporeRepublic of Singapore

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