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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 Chan
Original Paper

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.

Figure

Metabolic footprinting of tumorigenic and nontumorigenic uroepithelial cells using GCxGCTOFMS

Keywords

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

Notes

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)

References

  1. 1.
    Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ (2009) CA Cancer J Clin 59:225–249CrossRefGoogle Scholar
  2. 2.
    Lotan Y, Roehrborn CG (2002) J Urol 167:75–79CrossRefGoogle Scholar
  3. 3.
    Kell DB, Brown M, Davey HM, Dunn WB, Spasic I, Oliver SG (2005) Nat Rev Microbiol 3:557–565CrossRefGoogle Scholar
  4. 4.
    Mapelli V, Olsson L, Nielsen J (2008) Trends Biotechnol 26:490–497CrossRefGoogle Scholar
  5. 5.
    Kaderbhai NN, Broadhurst DI, Ellis DI, Goodacre R, Kell DB (2003) Comp Funct Genomics 4:376–391CrossRefGoogle Scholar
  6. 6.
    Fiehn O (2002) Plant Mol Biol 48:155–171CrossRefGoogle Scholar
  7. 7.
    Buchholz A, Hurlebaus J, Wandrey C, Takors R (2002) Biomol Eng 19:5–15CrossRefGoogle Scholar
  8. 8.
    Villas-Boas SG, Noel S, Lane GA, Attwood G, Cookson A (2006) Anal Biochem 349:297–305CrossRefGoogle Scholar
  9. 9.
    Abel CB, Lindon JC, Noble D, Rudd BA, Sidebottom PJ, Nicholson JK (1999) Anal Biochem 270:220–230CrossRefGoogle Scholar
  10. 10.
    Miccheli AT, Miccheli A, Di Clemente R, Valerio M, Coluccia P, Bizzarri M, Conti F (2006) Biochim Biophys Acta 1760:1723–1731Google Scholar
  11. 11.
    Dunn WB, Brown M, Worton SA, Crocker IP, Broadhurst D, Horgan R, Kenny LC, Baker PN, Kell DB, Heazell AE (2009) Placenta 30:974–980CrossRefGoogle Scholar
  12. 12.
    Pope GA, MacKenzie DA, Defernez M, Aroso MA, Fuller LJ, Mellon FA, Dunn WB, Brown M, Goodacre R, Kell DB, Marvin ME, Louis EJ, Roberts IN (2007) Yeast 24:667–679CrossRefGoogle Scholar
  13. 13.
    Pasikanti KK, Ho PC, Chan EC (2008) J Chromatogr B Analyt Technol Biomed Life Sci 871:202–211CrossRefGoogle Scholar
  14. 14.
    Want EJ, Nordstrom A, Morita H, Siuzdak G (2007) J Proteome Res 6:459–468CrossRefGoogle Scholar
  15. 15.
    Almstetter MF, Appel IJ, Gruber MA, Lottaz C, Timischl B, Spang R, Dettmer K, Oefner PJ (2009) Anal Chem 81:5731–5739CrossRefGoogle Scholar
  16. 16.
    Li X, Xu Z, Lu X, Yang X, Yin P, Kong H, Yu Y, Xu G (2009) Anal Chim Acta 633:257–262CrossRefGoogle Scholar
  17. 17.
    Ralston-Hooper K, Hopf A, Oh C, Zhang X, Adamec J, Sepulveda MS (2008) Aquat Toxicol 88:48–52CrossRefGoogle Scholar
  18. 18.
    Mohler RE, Dombek KM, Hoggard JC, Pierce KM, Young ET, Synovec RE (2007) Analyst 132:756–767CrossRefGoogle Scholar
  19. 19.
    Shellie RA, Welthagen W, Zrostlikova J, Spranger J, Ristow M, Fiehn O, Zimmermann R (2005) J Chromatogr A 1086:83–90CrossRefGoogle Scholar
  20. 20.
    Cortes HJ, Winniford B, Luong J, Pursch M (2009) J Sep Sci 32:883–904CrossRefGoogle Scholar
  21. 21.
    Ong RC, Marriott PJ (2002) J Chromatogr Sci 40:276–291Google Scholar
  22. 22.
    Welthagen W, Shellie RA, Spranger J, Ristow M, Zimmermann R, Fiehn O (2005) Metabolomics 1:65–73CrossRefGoogle Scholar
  23. 23.
    Kopka J, Schauer N, Krueger S, Birkemeyer C, Usadel B, Bergmuller E, Dormann P, Weckwerth W, Gibon Y, Stitt M, Willmitzer L, Fernie AR, Steinhauser D (2005) Bioinformatics 21:1635–1638CrossRefGoogle Scholar
  24. 24.
    Wishart DS, Tzur D, Knox C, Eisner R, Guo AC, Young N, Cheng D, Jewell K, Arndt D, Sawhney S, Fung C, Nikolai L, Lewis M, Coutouly MA, Forsythe I, Tang P, Shrivastava S, Jeroncic K, Stothard P, Amegbey G, Block D, Hau DD, Wagner J, Miniaci J, Clements M, Gebremedhin M, Guo N, Zhang Y, Duggan GE, Macinnis GD, Weljie AM, Dowlatabadi R, Bamforth F, Clive D, Greiner R, Li L, Marrie T, Sykes BD, Vogel HJ, Querengesser L (2007) Nucleic Acids Res 35:D521–D526CrossRefGoogle Scholar
  25. 25.
    Mendes P (2002) Brief Bioinform 3:134–145CrossRefGoogle Scholar
  26. 26.
    Pasikanti KK, Ho PC, Chan EC (2008) Rapid Commun Mass Spectrom 22:2984–2992CrossRefGoogle Scholar
  27. 27.
    Koek MM, Muilwijk B, van Stee LL, Hankemeier T (2008) J Chromatogr A 1186:420–429CrossRefGoogle Scholar
  28. 28.
    Pasikanti KK, Esuvaranathan K, Ho PC, Mahendran R, Kamaraj R, Wu QH, Chiong E, Chan EC (2010) J Proteome Res 9:2988–2995CrossRefGoogle Scholar
  29. 29.
    Dalluge J, Beens J, Brinkman UA (2003) J Chromatogr A 1000:69–108CrossRefGoogle Scholar
  30. 30.
    Donato P, Tranchida PQ, Dugo P, Dugo G, Mondello L (2007) J Sep Sci 30:508–526CrossRefGoogle Scholar
  31. 31.
    Ong R, Marriott P, Morrison P, Haglund P (2002) J Chromatogr A 962:135–152CrossRefGoogle Scholar
  32. 32.
    Begley P, Francis-McIntyre S, Dunn WB, Broadhurst DI, Halsall A, Tseng A, Knowles J, Goodacre R, Kell DB (2009) Anal Chem 81:7038–7046CrossRefGoogle Scholar
  33. 33.
    Oh C, Huang X, Regnier FE, Buck C, Zhang X (2008) J Chromatogr A 1179:205–215CrossRefGoogle Scholar
  34. 34.
    Holmes E, Antti H (2002) Analyst 127:1549–1557CrossRefGoogle Scholar
  35. 35.
    Wiklund S, Johansson E, Sjostrom L, Mellerowicz EJ, Edlund U, Shockcor JP, Gottfries J, Moritz T, Trygg J (2008) Anal Chem 80:115–122CrossRefGoogle Scholar
  36. 36.
    Trygg J, Holmes E, Lundstedt T (2007) J Proteome Res 6:469–479CrossRefGoogle Scholar
  37. 37.
    Theodoropoulos VE, Lazaris A, Sofras F, Gerzelis I, Tsoukala V, Ghikonti I, Manikas K, Kastriotis I (2004) Eur Urol 46:200–208CrossRefGoogle Scholar
  38. 38.
    Ioachim E, Michael M, Salmas M, Michael MM, Stavropoulos NE, Malamou-Mitsi V (2006) Urol Int 77:255–263CrossRefGoogle Scholar
  39. 39.
    Griffin JL, Shockcor JP (2004) Nat Rev Cancer 4:551–561CrossRefGoogle Scholar
  40. 40.
    Dang CV, Semenza GL (1999) Trends Biochem Sci 24:68–72CrossRefGoogle Scholar
  41. 41.
    Pedersen PL, Mathupala S, Rempel A, Geschwind JF, Ko YH (2002) Biochim Biophys Acta 1555:14–20CrossRefGoogle Scholar

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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