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In-gel digestion for mass spectrometric characterization of proteins and proteomes

Nature Protocols volume 1, pages 2856–2860 (2006)Cite this article

Abstract

In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.

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Figure 1: Characterization of a modified protein by GeLCMS.
Figure 2: Characterization of a complex proteome.

References

  1. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    Article  CAS  Google Scholar 

  2. Shevchenko, A., Loboda, A., Ens, W., Schraven, B. & Standing, K.G. Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry. Electrophoresis 22, 1194–1203 (2001).

    Article  CAS  Google Scholar 

  3. Havlis, J., Thomas, H., Sebela, M. & Shevchenko, A. Fast-response proteomics by accelerated in-gel digestion of proteins. Anal. Chem. 75, 1300–1306 (2003).

    Article  CAS  Google Scholar 

  4. Havlis, J. & Shevchenko, A. Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry. Anal. Chem. 76, 3029–3036 (2004).

    Article  CAS  Google Scholar 

  5. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    Article  CAS  Google Scholar 

  6. Yao, X., Freas, A., Ramirez, J., Demirev, P.A. & Fenselau, C. Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Anal. Chem. 73, 2836–2842 (2001).

    Article  CAS  Google Scholar 

  7. Shevchenko, A. & Shevchenko, A. Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry. Anal. Biochem. 296, 279–283 (2001).

    Article  CAS  Google Scholar 

  8. Mason, C.J. et al. A method for automatically interpreting mass spectra of 18O labeled isotopic clusters. Mol. Cell Proteomics in the press (2006).

  9. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell Proteomics 1, 376–386 (2002).

    Article  CAS  Google Scholar 

  10. Ong, S.-E. & Mann, M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat. Protocols 1, 2650–2660 (2006).

    Article  CAS  Google Scholar 

  11. Shevchenko, A., Chernushevich, I., Wilm, M. & Mann, M. De novo peptide sequencing by nanoelectrospray tandem mass spectrometry using triple quadrupole and quadrupole/time-of-flight instruments. Methods Mol. Biol. 146, 1–16 (2000).

    CAS  PubMed  Google Scholar 

  12. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    Article  CAS  Google Scholar 

  13. Shevchenko, A. et al. Rapid “de novo” peptide sequencing by a combination of nanoelectrospray, isotopic labelling and a quadrupole/time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 11, 1015–1024 (1997).

    Article  CAS  Google Scholar 

  14. Shevchenko, A., Sunyaev, S., Liska, A. & Bork, P. Nanoelectrospray tandem mass spectrometry and sequence similarity searching for identification of proteins from organisms with unknown genomes. Methods Mol. Biol. 211, 221–234 (2003).

    CAS  PubMed  Google Scholar 

  15. Thomas, H., Havlis, J., Peychl, J. & Shevchenko, A. Dried-droplet probe preparation on AnchorChip targets for navigating the acquisition of matrix-assisted laser desorption/ionization time-of-flight spectra by fluorescence of matrix/analyte crystals. Rapid Commun. Mass Spectrom. 18, 923–930 (2004).

    Article  CAS  Google Scholar 

  16. Wielsch, N. et al. Rapid validation of protein identifications with the borderline statistical confidence via de novo sequencing and MS BLAST searches. J. Proteome Res. 5, 2448–2456 (2006).

    Article  CAS  Google Scholar 

  17. Pichler, A. et al. SUMO modification of the ubiquitin-conjugating enzyme E2-25K. Nat. Struct. Mol. Biol. 12, 264–269 (2005).

    Article  CAS  Google Scholar 

  18. Ong, S.E. & Mann, M. Mass spectrometry-based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–262 (2005).

    Article  CAS  Google Scholar 

  19. de Godoy, L.M. et al. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. Genome Biol. 7, R50 (2006).

    Article  Google Scholar 

  20. Mitulovic, G. et al. An improved method for tracking and reducing the void volume in nano HPLC-MS with micro trapping columns. Anal. Bioanal. Chem. 376, 946–951 (2003).

    Article  CAS  Google Scholar 

  21. Sebela, M. et al. Thermostable trypsin conjugates for high-throughput proteomics: synthesis and performance evaluation. Proteomics 6, 2959–2963 (2006).

    Article  CAS  Google Scholar 

  22. Olsen, J.V. & Mann, M. Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation. Proc. Natl. Acad. Sci. USA 101, 13417–13422 (2004).

    Article  CAS  Google Scholar 

  23. Steen, H. & Mann, M. The abc's (and xyz's) of peptide sequencing. Nat. Rev. Mol. Cell Biol. 5, 699–711 (2004).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, 01307, Germany

    Andrej Shevchenko, Henrik Tomas & Jan Havli

  2. Max Planck Institute for Biochemistry, Am Klopferspitz 18, Martinsried, 82152, Germany

    Jesper V Olsen & Matthias Mann

Authors
  1. Andrej Shevchenko
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  2. Henrik Tomas
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  3. Jan Havli
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  4. Jesper V Olsen
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Correspondence to Andrej Shevchenko or Matthias Mann.

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Shevchenko, A., Tomas, H., Havli, J. et al. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1, 2856–2860 (2006). https://doi.org/10.1038/nprot.2006.468

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