Toward a high-throughput approach to quantitative proteomic analysis: Expression-dependent protein identification by mass spectrometry

  • Timothy J. GriffinEmail author
  • David K. M. Han
  • Steven P. Gygi
  • Beate Rist
  • Hookeun Lee
  • Ruedi Aebersold
  • Kenneth C. Parker


The isotope-coded affinity tag (ICAT) [1] technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.


Peptide Mass Spectro Electrospray Ionization Tandem Mass Spectrometry HFBA Quantitative Proteomic Analysis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994–999.CrossRefGoogle Scholar
  2. 2.
    Golub, T. R.; Slonim, D. K.; Tamayo, P.; Huard, C.; Gaasenbeek, M.; Mesirov, J. P.; Coller, H.; Loh, M. L.; Downing, J. R.; Caligiuri, M. A.; Bloomfield, C. D.; Lander, E. S. Science 1999, 286, 531–537.CrossRefGoogle Scholar
  3. 3.
    Alizadeh, A. A.; Eisen, M. B.; Davis, R. E.; Ma, C.; Lossos, I. S.; Rosenwald, A.; Boldrick, J. C.; Sabet, H.; Tran, T.; Yu, X.; Powell, J. I.; Yang, L.; Marti, G. E.; Moore, T.; Hudson, J., Jr.; Lu, L.; Lewis, D. B.; Tibshirani, R.; Sherlock, G.; Chan, W. C.; Greiner, T. C.; Weisenburger, D. D.; Armitage, J. O.; Warnke, R.; Staudt, L. M., et al. Nature 2000, 403, 503–511.CrossRefGoogle Scholar
  4. 4.
    Scherf, U.; Ross, D. T.; Waltham, M.; Smith, L. H.; Lee, J. K.; Tanabe, L.; Kohn, K. W.; Reinhold, W. C.; Myers, T. G.; Andrews, D. T.; Scudiero, D. A.; Eisen, M. B.; Sausville, E. A.; Pommier, Y.; Botstein, D.; Brown, P. O.; Weinstein, J. N. Nat. Genet. 2000, 24, 236–244.CrossRefGoogle Scholar
  5. 5.
    Ross, D. T.; Scherf, U.; Eisen, M. B.; Perou, C. M.; Rees, C.; Spellman, P.; Iyer, V.; Jeffrey, S. S.; Van de Rijn, M.; Waltham, M.; Pergamenschikov, A.; Lee, J. C.; Lashkari, D.; Shalon, D.; Myers, T. G.; Weinstein, J. N.; Botstein, D.; Brown, P. O. Nat. Genet. 2000, 24, 227–235.CrossRefGoogle Scholar
  6. 6.
    Lockhart, D. J.; Winzeler, E. A. Nature 2000, 405, 827–836.CrossRefGoogle Scholar
  7. 7.
    Lashkari, D. A.; DeRisi, J. L.; McCusker, J. H.; Namath, A. F.; Gentile, C.; Hwang, S. Y.; Brown, P. O.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13057–13062.CrossRefGoogle Scholar
  8. 8.
    Marton, M. J.; DeRisi, J. L.; Bennett, H. A.; Iyer, V. R.; Meyer, M. R.; Roberts, C. J.; Stoughton, R.; Burchard, J.; Slade, D.; Dai, H.; Bassett, D. E., Jr.; Hartwell, L. H.; Brown, P. O.; Friend, S. H. Nat. Med. 1998, 4, 1293–1301.CrossRefGoogle Scholar
  9. 9.
    Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol. Cell Biol. 1999, 19, 1720–1730.Google Scholar
  10. 10.
    Yates, J. R., 3rd. Trends Genet. 2000, 16, 5–8.CrossRefGoogle Scholar
  11. 11.
    Pandey, A.; Mann, M. Nature 2000, 405, 837–846.CrossRefGoogle Scholar
  12. 12.
    Gygi, S. P.; Aebersold, R. Curr. Opin. Chem. Biol. 2000, 4, 489–494.CrossRefGoogle Scholar
  13. 13.
    Washburn, M. P.; Wolters, D.; Yates, J. R., 3rd. Nat. Biotechnol. 2001, 19, 242–247.CrossRefGoogle Scholar
  14. 14.
    Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591–6596.CrossRefGoogle Scholar
  15. 15.
    Pasa-Tolic, L.; Jensen, P.; Anderson, G.; Lipton, M.; Peden, K.; Martinovic, S.; Tolic, N.; Bruce, J.; Smith, R. J. Am. Chem. Soc. 1999, 121, 7949–7950.CrossRefGoogle Scholar
  16. 16.
    Geng, M.; Ji, J.; Regnier, F. E. J. Chromatogr. A 2000, 870, 295–313.CrossRefGoogle Scholar
  17. 17.
    Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047–4057.CrossRefGoogle Scholar
  18. 18.
    Gygi, S. P.; Aebersold, R. In Proteomics: A Trends Guide; Mann, M.; Blackstock, W., Eds.; Elsevier: London, 2000; p 32.Google Scholar
  19. 19.
    Deterding, L. J.; Moseley, M. A.; Tomer, K. B.; Jorgenson, J. W. J. Chromatogr. 1991, 554, 73–82.CrossRefGoogle Scholar
  20. 20.
    Hunt, D. F.; Yates, J. R. D.; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233–6237.CrossRefGoogle Scholar
  21. 21.
    Papayannopoupos, I. A. Mass Spectrom. Reviews 1995, 14, 49–73.CrossRefGoogle Scholar
  22. 22.
    Shabanowitz, J.; Settlage, R. E.; Marto, J. A.; Christian, R. E.; White, F. M.; Russo, P. S.; Martin, S. E.; Hunt, D. F. In Mass Spectrometry in Biology and Medicine; Burlingame, A. L.; Carr, S. A.; Baldwin, M. A., Eds.; Humana Press: Totowa, 2000; p 163.Google Scholar
  23. 23.
    Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C.; Yates, J. R., 3rd. Anal. Chem. 2000, 72, 757–763.CrossRefGoogle Scholar
  24. 24.
    Figeys, D.; Aebersold, R. Electrophoresis 1997, 18, 360–368.CrossRefGoogle Scholar
  25. 25.
    Courchesne, P. L.; Jones, M. D.; Robinson, J. H.; Spahr, C. S.; McCracken, S.; Bentley, D. L.; Luethy, R.; Patterson, S. D. Electrophoresis 1998, 19, 956–967.CrossRefGoogle Scholar
  26. 26.
    Eng, J.; McCormack, A. L.; Yates, J. R., 3rd. J. Am. Soc. Mass Spectrom. 1994, 5, 976–989.CrossRefGoogle Scholar
  27. 27.
    Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R. S.; Hoyes, J.; Bateman, R. H. Rapid Commun. Mass Spectrom. 1996, 10, 889–896.CrossRefGoogle Scholar
  28. 28.
    Shevchenko, A.; Chernushevich, I.; Ens, W.; Standing, K. G.; Thomson, B.; Wilm, M.; Mann, M. Rapid Commun. Mass Spectrom. 1997, 11, 1015–1024.CrossRefGoogle Scholar
  29. 29.
    Bae, V. L.; Jackson-Cook, C. K.; Brothman, A. R.; Maygarden, S. J.; Ware, J. L. Int. J. Cancer 1994, 58, 721–729.CrossRefGoogle Scholar
  30. 30.
    Rossi, M. C.; Zetter, B. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6197–6201.CrossRefGoogle Scholar
  31. 31.
    Clauser, K. R.; Baker, P.; Burlingame, A. L. Anal. Chem. 1999, 71, 2871–2882.CrossRefGoogle Scholar
  32. 32.
    Fenyo, D.; Qin, J.; Chait, B. T. Electrophoresis 1998, 19, 998–1005.CrossRefGoogle Scholar
  33. 33.
    Thomas, R.; True, L. D.; Bassuk, J. A.; Lange, P. H.; Vessella, R. L. Clin. Cancer Res. 2000, 6, 1140–1149.Google Scholar
  34. 34.
    Motamed, K. Int. J. Biochem. Cell Biol. 1999, 31, 1363–1366.CrossRefGoogle Scholar
  35. 35.
    Baskies, A. M.; Chretien, P. B.; Weiss, J. F.; Makuch, R. W.; Beveridge, R. A.; Catalona, W. J.; Spiegel, H. E. Cancer 1980, 45, 3050–3060.CrossRefGoogle Scholar
  36. 36.
    Wong, I. H.; Lau, W. Y.; Leung, T.; Johnson, P. J. Cancer Lett. 2000, 156, 141–149.CrossRefGoogle Scholar
  37. 37.
    Han, D. K. M.; Eng, J.; Zhou, H.; Aebersold, R. Nature Biotechnology, in press.Google Scholar
  38. 38.
    Griffin, T. J.; Loboda, A.; Gygi, S. P.; Rist, B.; Jilkine, A.; Ens, W.; Standing, K. G.; Aebersold, R. Anal. Chem. 2001, 73, 978–986.CrossRefGoogle Scholar
  39. 39.
    Goodlett, D. R.; Bruce, J. E.; Anderson, G. A.; Rist, B.; Pasa-Tolic, L.; Fiehn, O.; Smith, R. D.; Aebersold, R. Anal. Chem. 2000, 72, 1112–1118.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2001

Authors and Affiliations

  • Timothy J. Griffin
    • 1
    Email author
  • David K. M. Han
    • 1
  • Steven P. Gygi
    • 1
  • Beate Rist
    • 1
  • Hookeun Lee
    • 1
  • Ruedi Aebersold
    • 1
  • Kenneth C. Parker
    • 2
  1. 1.Department of Molecular BiotechnologyUniversity of WashingtonSeattleUSA
  2. 2.Applied BiosystemsFraminghamUSA

Personalised recommendations