Nanoelectrospray Tandem Mass Spectrometry and Sequence Similarity Searching for Identification of Proteins from Organisms with Unknown Genomes

  • Anna Shevchenko
  • Shamil Sunyaev
  • Adam Liska
  • Peer Bork
  • Andrej Shevchenko
Part of the Methods in Molecular Biology™ book series (MIMB, volume 211)


Recent developments in technology and instrumentation have made mass spectrometry the method of choice for identification of proteins in a sequence database [reviewed in refs. (1, 2, 3, 4)].Regardless of what mass spectrometric technology is applied, it is ultimately required that acquired mass spectra be accurately matched to a protein sequence from the corresponding database entry.


  1. 1.
    Chalmers, M. J. and Gaskell, S. J. (2000) Advances in mass spectrometry for proteome analysis. Curr. Opin. Biotechnol. 11, 384–390.PubMedCrossRefGoogle Scholar
  2. 2.
    Lahm, H. W. and Langen, H. (2000) Mass spectrometry: a tool for the identification of proteins separated by gels. Electrophoresis 21, 2105–2114.PubMedCrossRefGoogle Scholar
  3. 3.
    Pandey, A. and Mann, M. (2000) Proteomics to study genes and genomes. Nature 405, 837–846.PubMedCrossRefGoogle Scholar
  4. 4.
    Mann, M., Hendrickson, R. C., and Pandey, A. (2001) Analysis of proteins and proteomes by mass spectrometry. Annu. Rev. Biochem. 70, 437–473.PubMedCrossRefGoogle Scholar
  5. 5.
    Podtelejnikov, A. V., Bachi, A., and Mann, M. (1998) Cross-species identification of proteins by high mass accuracy MALDI peptide mapping, in Proceedings 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, Abstract no. 212.Google Scholar
  6. 6.
    Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., et al. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nanoelectrospray mass spectrometry. Nature 379, 466–469.PubMedCrossRefGoogle Scholar
  7. 7.
    Shevchenko, A., Wilm, M., and Mann, M. (1997) Peptide sequencing by mass spectrometry for homology searches and cloning of genes. J. Protein Chem. 16, 481–90.PubMedCrossRefGoogle Scholar
  8. 8.
    Roepstorff, P. and Fohlman, J. (1984) Proposed nomenclature for sequence ions. Biomed. Mass Spectrom. 11, 601.PubMedCrossRefGoogle Scholar
  9. 9.
    Mann, M. and Wilm, M. (1994) Error tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390–399.PubMedCrossRefGoogle Scholar
  10. 10.
    Hunt, D. F., Yates, J. R., Shabanowitz, J., Winston, S., and Hauer, C. R. (1986) Peptide sequencing by tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 83, 6233–6237.PubMedCrossRefGoogle Scholar
  11. 11.
    Shevchenko, A., Chernushevich, I., Ens, W., Standing, K. G., Thomson, B., Wilm, M., et al. (1997) Rapid’ De Novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 11, 1015–1024.PubMedCrossRefGoogle Scholar
  12. 12.
    McNagny, K. M., Petterson, I., Rossi, F., Flamme, I., Shevchenko, A., Mann, M., et al. (1997) Thrombomucin, a novel cell surface protein that defines thromb-ocytes and multipotent hematopoetic progenitors.J. CellBiol. 138, 1395–1407.CrossRefGoogle Scholar
  13. 13.
    Lingner, J., Hughes, T. R., Shevchenko, A., Mann, M., Lundblad, V., and Cech, T. R. (1997) Reverse transcriptase motifs in the catalytic subunits of telomerase. Science 276, 561–567.PubMedCrossRefGoogle Scholar
  14. 14.
    Chen, R. H., Shevchenko, A., Mann, M., and Murray, A. W. (1998) Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores. J. Cell Biol. 143, 283–295.PubMedCrossRefGoogle Scholar
  15. 15.
    Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., Rourke, K. O., Shevchenko, A., Jian N., et al. (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817–827.PubMedCrossRefGoogle Scholar
  16. 16.
    Aigner, S., Lingner, J., Goodrich, K. J., Grosshans, C. A., Shevchenko, A., Mann, M., et al. (2000) Euplotes telomerase contains an La motif protein produced by apparent translational frameshifting. EMBO J. 19, 6230–6239.PubMedCrossRefGoogle Scholar
  17. 17.
    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
  18. 18.
    Huang, L., Jacob, R. J., Pegg, S. C., Baldwin, M. A., Wang, C. C., Burlingame, A. A., et al. (2001) Functional assignment of the 20S proteasome from Trypanosoma brucei using mass spectrometry and new bioinformatics approaches. J. Biol. Chem. 17, 28327–28339.CrossRefGoogle Scholar
  19. 19.
    Shevchenko, A., Sunyaev, S., Loboda, A., Shevchenko, A., Bork, P., Ens, W., et al. (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI-Quadrupole Time-of-Flight mass spectrometry and BLAST homology searching. Anal. Chem. 73, 1917–1926.PubMedCrossRefGoogle Scholar
  20. 20.
    Johnson, R. S. and Taylor, J. A. (2000) Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry. Methods Mol. Biol. 146, 41–61.PubMedGoogle Scholar
  21. 21.
    Shevchenko, A., Loboda, A., Shevchenko, A., Ens, W., and Standing, K. G. (2000) MALDI Quadrupole Time-of-Flight mass spectrometry: a powerful tool for proteomic research. Anal. Chem. 72, 2132–2141.PubMedCrossRefGoogle Scholar
  22. 22.
    Medzihradszky, K. F., Campbell, J. M., Baldwin, M. A., Falick, A. M., Juhasz, P., Vestal, M. L., et al. (2000) The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer. Anal. Chem. 72, 552–558.PubMedCrossRefGoogle Scholar
  23. 23.
    Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682.PubMedCrossRefGoogle Scholar
  24. 24.
    Wilm, M. and Mann, M. (1996) Analytical properties of the nano electrospray ion source. Anal. Chem. 66, 1–8.CrossRefGoogle Scholar
  25. 25.
    Jensen, O. N., Wilm, M., Shevchenko, A., and Mann, M. (1999) Peptide sequencing of 2-DE gel-isolated proteins by nanoelectrospray tandem mass spectrometry. Methods Mol. Biol. 112, 571–588.PubMedGoogle Scholar
  26. 26.
    Shevchenko, A., Chernushevich, I., and Mann, M. (1998) High sensitivity analysis of gel separated proteins by a quadrupole-TOF tandem mass spectrometer, in Proceedings 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, Abstract no. 237.Google Scholar
  27. 27.
    Altschul, S., Madden, T., Schaffer, A., Zhang, J., Zhang, Z., Miller, W., et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.PubMedCrossRefGoogle Scholar
  28. 28.
    Tournebize, R., Popov, A., Kinoshita, K., Ashford, A. J., Rybina, S., Pozniakovsky, A., et al. (2000) Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts. Nat. Cell Biol. 2, 13–19.PubMedCrossRefGoogle Scholar
  29. 29.
    Taylor, J. A. and Johnson, R. S. (2001) Implementation and uses of automated de novo peptide sequencing by tandem mass spectrometry. Anal. Chem. 73, 2594–2604.PubMedCrossRefGoogle Scholar
  30. 30.
    Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.PubMedCrossRefGoogle Scholar
  31. 31.
    Shevchenko, A., Chernushevich, I., Wilm, M., and Mann, M. (2000) Methods Mol. Biol. 146, 1–16.PubMedGoogle Scholar
  32. 32.
    Shevchenko, A. (2001) Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry. Anal. Biochem. 296, 279–283.PubMedCrossRefGoogle Scholar
  33. 33.
    Wilm, M., Neubauer, G., and Mann, M. (1996) Parent ion scans of unseparated peptide mixtures. Anal. Chem. 68, 527–533.PubMedCrossRefGoogle Scholar
  34. 34.
    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
  35. 35.
    Altschul, S. F., Boguski, M. S., Gish, W., and Wootton, J. C. (1994) Issues in searching molecular sequence databases. Nat. Genet. 6, 119–129.PubMedCrossRefGoogle Scholar
  36. 36.
    Gaeta, B. A. (2000) BLAST on the Web. Biotechniques 28, 436–440.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Anna Shevchenko
    • 1
  • Shamil Sunyaev
    • 2
  • Adam Liska
    • 1
  • Peer Bork
    • 2
  • Andrej Shevchenko
    • 2
  1. 1.European Molecular Biology LaboratoryHeidelbergGermany
  2. 2.MPI of Molecular Cell Biology and GeneticsDresdenGermany

Personalised recommendations