Advertisement

A Protocol for Top-Down Proteomics Using HPLC and ETD/PTR-MS

  • Sarah R. Hart
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 658)

Abstract

Analysis of intact proteins by tandem mass spectrometry has mostly been confined to high-end mass spectrometry platforms. This protocol describes the application of routine HPLC to separate proteins, MALDI-ToF mass spectrometry to interrogate intact protein species and electron transfer dissociation/proton transfer reaction within a quadrupole ion trap to perform tandem mass spectrometry.

Key words

Mass spectrometry intact protein electron transfer dissociation proton transfer reaction high-performance liquid chromatography 

Notes

Acknowledgements

I would like to thank current and former members of the Michael Barber Centre, University of Manchester for constructive comments during this work. John Cottrell (Matrix Sciences) provided demonstration licensing of top-down Mascot. Ken Cook (Dionex) provided demonstration of HPLC columns and assisted in setting up gradients. Carsten Baessmann, Andrea Kiehne, Markus Lubeck, Andrea Schneider and Julia Smith (Bruker Daltonics) have provided essential guidance both with ETD/PTR experiments and with data processing.

References

  1. 1.
    Cravatt, B. F., Simon, G. M., and Yates, J. R., 3rd (2007) The biological impact of mass-spectrometry-based proteomics. Nature 450, 991–1000.PubMedCrossRefGoogle Scholar
  2. 2.
    Hart, S. R., and Gaskell, S. J. (2005) LC-tandem MS in proteome characterization. TrAC Trends Anal. Chem. 24, 566–75.CrossRefGoogle Scholar
  3. 3.
    Hart, S. R., and Gaskell, S. J. (2008) Methods of proteome analysis: challenges and opportunities. SEB Exp. Biol. Ser. 61, 37–64.PubMedGoogle Scholar
  4. 4.
    Aebersold, R., and Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422, 198–207.PubMedCrossRefGoogle Scholar
  5. 5.
    Li, G., Waltham, M., Anderson, N. L., Unsworth, E., Treston, A., and Weinstein, J. N. (1997) Rapid mass spectrometric identification of proteins from two-dimensional polyacrylamide gels after in gel proteolytic digestion. Electrophoresis 18, 391–402.PubMedCrossRefGoogle Scholar
  6. 6.
    Washburn, M. P., Wolters, D., and Yates, J. R., 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.PubMedCrossRefGoogle Scholar
  7. 7.
    Yates, J. R., 3rd, Carmack, E., Hays, L., Link, A. J., and Eng, J. K. (1999) Automated protein identification using microcolumn liquid chromatography-tandem mass spectrometry. Methods Mol. Biol. 112, 553–569.PubMedGoogle Scholar
  8. 8.
    Zhen, Y., Xu, N., Richardson, B., Becklin, R., Savage, J. R., Blake, K., et al. (2004) Development of an LC-MALDI method for the analysis of protein complexes. J. Am. Soc. Mass Spectrom. 15, 803–822.PubMedCrossRefGoogle Scholar
  9. 9.
    Siuti, N., and Kelleher, N. L. (2007) Decoding protein modifications using top-down mass spectrometry. Nat. Methods 4, 817–21.PubMedCrossRefGoogle Scholar
  10. 10.
    Kelleher, N. L. (2004) Top-down proteomics. Anal. Chem. 76, 197A-203A.PubMedCrossRefGoogle Scholar
  11. 11.
    Garcia, B. A., Shabanowitz, J., and Hunt, D. F. (2005) Analysis of protein phosphorylation by mass spectrometry. Methods 35, 256–264.PubMedCrossRefGoogle Scholar
  12. 12.
    Wiesner, J., Premsler, T., and Sickmann, A. (2008) Application of electron transfer dissociation (ETD) for the analysis of posttranslational modifications. Proteomics 8, 4466–4483.PubMedCrossRefGoogle Scholar
  13. 13.
    Thingholm, T. E., Jorgensen, T. J., Jensen, O. N., and Larsen, M. R. (2006) Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat. Protoc. 1, 1929–1935.PubMedCrossRefGoogle Scholar
  14. 14.
    Burlingame, A. L., Zhang, X., and Chalkley, R. J. (2005) Mass spectrometric analysis of histone posttranslational modifications. Methods 36, 383–394.PubMedCrossRefGoogle Scholar
  15. 15.
    Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., Yoshida, T., Matsuo, T. (1988) Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2, 151–153.Google Scholar
  16. 16.
    Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64–71.PubMedCrossRefGoogle Scholar
  17. 17.
    Zubarev, R. A., Kelleher, N. L., and McLafferty, F. W. (1998) Electron capture dissociation of multiply charged protein cations. A nonergodic process. J. Am. Chem. Soc. 120, 3265–3266.CrossRefGoogle Scholar
  18. 18.
    Lifshitz, C. (2006) Intramolecular vibrational energy redistribution and ergodicity of biomolecular dissociation. In “Principles of mass spectrometry applied to biomolecules,” Laskin, J., Lifshitz, Chava (Eds.). Wiley, Hoboken, New Jersey, pp. 239–75.CrossRefGoogle Scholar
  19. 19.
    Syka, J. E., Coon, J. J., Schroeder, M. J., Shabanowitz, J., and Hunt, D. F. (2004) Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. USA 101, 9528–33.PubMedCrossRefGoogle Scholar
  20. 20.
    Coon, J. J., Ueberheide, B., Syka, J. E., Dryhurst, D. D., Ausio, J., Shabanowitz, J., et al. (2005) Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 102, 9463–9468.PubMedCrossRefGoogle Scholar
  21. 21.
    Kaplan, D. A., Hartmer, R., Speir, J. P., Stoermer, C., Gumerov, D., Easterling, M. L., et al. (2008) Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer. Rapid Commun. Mass Spectrom. 22, 271–278.PubMedCrossRefGoogle Scholar
  22. 22.
    McAlister, G. C., Berggren, W. T., Griep-Raming, J., Horning, S., Makarov, A., Phanstiel, D., et al. (2008) A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-orbitrap mass spectrometer. J. Proteome Res. 7, 3127–3136.PubMedCrossRefGoogle Scholar
  23. 23.
    Hartmer, R., Lubeck, M., Bäßmann, C., Brekenfeld, A. (2007) New setup for top-down characterization of proteins via consecutive ion/ion reactions. J. Biomol. Tech. 18, 34–35.Google Scholar
  24. 24.
    Anderson, N. L., and Anderson, N. G. (2002) The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell Proteomics 1, 845–867.PubMedCrossRefGoogle Scholar
  25. 25.
    Liu, T., Qian, W. J., Gritsenko, M. A., Xiao, W., Moldawer, L. L., Kaushal, A., et al. (2006) High dynamic range characterization of the trauma patient plasma proteome. Mol. Cell Proteomics 5, 1899–1913.PubMedCrossRefGoogle Scholar
  26. 26.
    Westerlund, B. (2004) Ion–exchange chromatography. In “Purifying proteins for proteomics: a laboratory manual,” Simpson, R. J., (Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 121–146Google Scholar
  27. 27.
    Gaskell, S. J. (1997) Electrospray: principles and practice. J Mass Spectrom 32, 677–688.Google Scholar
  28. 28.
    McLafferty, F. W., Horn, D. M., Breuker, K., Ge, Y., Lewis, M. A., Cerda, B., Zubarev, R. A., and Carpenter, B. K. (2001) Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance. J. Am. Soc. Mass Spectrom. 12, 245–249.Google Scholar
  29. 29.
    Samgina, T. Y., Artemenko, K. A., Gorshkov, V. A., Lebedev, A. T., Nielsen, M. L., Savistski, M. L., et al. (2007) Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges. Eur. J. Mass Spectrom. (Chichester, Eng) 13, 155–163.CrossRefGoogle Scholar
  30. 30.
    Lundgren, D. H., Han, D. K., and Eng, J. K. (2005) Protein identification using TurboSEQUEST. Curr Protoc Bioinformatics. Chapter 13, Unit 13 3.Google Scholar
  31. 31.
    Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567.PubMedCrossRefGoogle Scholar
  32. 32.
    Leduc, R. D., and Kelleher, N. L. (2007) Using ProSight PTM and related tools for targeted protein identification and characterization with high mass accuracy tandem MS data. Curr. Protoc. Bioinformatics Chapter 13, Unit 13 6.Google Scholar
  33. 33.
    Taylor, J. A., and Johnson, R. S. (1997) Sequence database searches via de novo peptide sequencing by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 11, 1067–1075.PubMedCrossRefGoogle Scholar
  34. 34.
    Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S., and Madden, T. L. (2008) NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9.PubMedCrossRefGoogle Scholar
  35. 35.
    Han, X., Jin, M., Breuker, K., and McLafferty, F. W. (2006) Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons. Science 314, 109–112.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Sarah R. Hart
    • 1
  1. 1.Michael Barber Centre for Mass SpectrometryUniversity of ManchesterManchesterUK

Personalised recommendations