Skip to main content
SpringerLink
Log in

Sample Preparation Methods for Mass Spectrometric Peptide Mapping Directly from 2-DE Gels

  • Protocol
2-D Proteome Analysis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 112))

Abstract

Polyacrylamide gel electrophoresis (PAGE) is probably the most general and widespread protein separation technique. This simple method is used in almost every molecular biology and biochemistry laboratory to monitor protein experiments, including consecutive stages of protein purification. PAGE may even be the final step of a purification protocol. Over the last decade, two-dimensional gel electrophoresis (2-D-PAGE) (1) has been developed, and applied to the separation of crude protein mixtures from organelles, tissues, and whole organisms (2).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.

    Google Scholar 

  2. Celis, J. E. (1995) The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways. Electrophoresis 16, 2177–2240.

    Article  PubMed  CAS  Google Scholar 

  3. Karas, M., Bachmann, D., Bahr, U., and Hillenkamp, F. (1987) Matrix-assisted ultraviolet laser desorption of non-volatile compounds. Int. J. Mass Spectrom. Ion Proc. 78, 53–68.

    Article  CAS  Google Scholar 

  4. Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T. (1991) Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal. Chem. 63, 1193A–1202A.

    Article  PubMed  CAS  Google Scholar 

  5. Beavis, R. C. and Chait, B. T. (1996) Matrix-assisted laser desorption mass spectrometry of proteins, in Methods in Enzymology (Karger, B. L. and Hancock, W. S., eds.), Academic, San Diego, pp. 519–551.

    Google Scholar 

  6. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989) Electrospray ionization for the mass spectrometry of large biomolecules. Science 246, 64–71.

    Article  PubMed  CAS  Google Scholar 

  7. Mann, M., and Wilm, M. (1995) Electrospray mass spectrometry for protein characterisation. Trends Biochem. Sci. 20, 219–223.

    Article  PubMed  CAS  Google Scholar 

  8. Chait, B. T., and Kent, S. B. H. (1992) Weighing naked proteins: Practical, high-accuracy mass measurement of peptides and proteins. Science 257, 1885–1894.

    Article  PubMed  CAS  Google Scholar 

  9. Siuzdak, G. (1994) The emergence of mass spectrometry in biochemical research. Proc. Natl. Acad. Sci. USA 91, 11,290–11,297.

    Article  PubMed  CAS  Google Scholar 

  10. Andersen, J. S., Svensson, B., and Roepstorff, P. (1996) Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry: Powerful analytical tools in recombinant protein chemistry. Nature Biotechnol. 14, 449–457.

    Article  CAS  Google Scholar 

  11. Mann, M. and Talbo, G. (1996) Developments in matrix-assisted laser desorption/ionization peptide mass spectrometry. Curr. Opinion Biotechnol. 7, 11–19.

    Article  CAS  Google Scholar 

  12. Roepstorff, P. (1997) Mass spectrometry in protein studies from genome to function. Curr. Opinion Biotechnol. 8, 6–13.

    Article  CAS  Google Scholar 

  13. Jensen, O. N., Shevchenko, A., and Mann, M. (1997) Protein analysis by mass spectrometry, in Protein Structure—A Practical Approach, 2nd ed. (Creighton, T. E., ed. IRL, Oxford University Press, Oxford, pp. 29–57.

    Google Scholar 

  14. Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., et al. (1996) Linking genome and proteome by mass spectrometry: large scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. USA 93, 14,440–14,445.

    Article  PubMed  CAS  Google Scholar 

  15. Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E., and Kent, S. B. H. (1987) Internal amino acid sequence analysis of proteins separated by two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. USA 84, 6970–6974.

    Article  PubMed  CAS  Google Scholar 

  16. Tempst, P., Link, A., Riviere, L., Fleming, M., and Elicone, C. (1990) Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: improved methods, applications and gene cloning strategies. Electrophoresis 11, 537–553.

    Article  PubMed  CAS  Google Scholar 

  17. Strupat, K., Karas, M., Hillenkamp, F., Eckerskorn, C., and Lottspeich, F. (1994) Matrix-assisted laser desorption ionization mass spectrometry of proteins electroblotted after polyacrylamide gel electrophoresis. Anal. Chem. 66, 464–470.

    Article  CAS  Google Scholar 

  18. Vestling, M. M., and Fenselau, C. (1994) Poly(vinylidene difluoride) membranes as the interface between laser desorption mass spectrometry, gel electrophoresis, and in situ proteolysis. Anal. Chem. 66, 471–477.

    Article  CAS  Google Scholar 

  19. Ogorzalek-Loo, R., Mitchell, C., Stevenson, T., Martin, S., Hines, W., Juhasz, P., et al. (1997) Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mapping. Electrophoresis 18, 382–390.

    Article  PubMed  CAS  Google Scholar 

  20. Schuhmacher, M., Glocker, M. O., Wunderlin, M., and Przybylski, M. (1996) Direct isolation of proteins from SDS-PAGE and analysis by ES MS. Electrophoresis 17, 848–854.

    Article  PubMed  CAS  Google Scholar 

  21. Cohen, S. and Chait, B. (1997) Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Anal. Biochem. 247, 257–267.

    Article  PubMed  CAS  Google Scholar 

  22. Zhang, W., Czernik, A., Yungwirth, T., Aebersold, R., and Chait, B. T. (1994) Matrix-assisted laser desorption mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis: determination of phosphorylation in synapsin I. Protein Sci. 3, 677–686.

    Article  PubMed  CAS  Google Scholar 

  23. Sutton, C. W., Pemberton, K. S., Cottrell, J. S., Wheeler, C. H., Dunn, M. J., and Pappin, D. J. (1995) Identification of myocardial proteins from two-dimensional gels by peptide mass fingerprinting. Electrophoresis 16, 308–316.

    Article  PubMed  CAS  Google Scholar 

  24. Covey, T. R., Huang, E. C., and Henion, J. D. (1991) Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions. Anal. Chem. 63, 1193–1200.

    Article  PubMed  CAS  Google Scholar 

  25. Griffin, P. R., Coffman, J. A., Hood, L. E., and Yates, J. R. III (1991) Structural analysis of proteins by capillary HPLC electrospray tandem mass spectrometry. Int. J. Mass Spectrom. Ion Proc. 111, 131–149.

    Article  CAS  Google Scholar 

  26. Hess, D., Covey, T. C., Winz, R., Brownsey, R., and Aebersold, R. (1993) Analytical and micropreparative peptide mapping by HPLC-electrospray mass spectrometry of proteins purified by gel electrophoresis. Protein Sci. 2, 1342–1351.

    Article  PubMed  CAS  Google Scholar 

  27. Figeys, D., Ducret, A., Yates, J. R., and Aebersold, R. (1996) Protein identification by solid phase microextraction capillary zone electrophoresis microelectrospray mass spectrometry. Nature Biotechnol. 14, 1579–1583.

    Article  CAS  Google Scholar 

  28. Fernandez, J., Andrews, L., and Mische, S. M. (1994) An improved procedure for enzymatic digestion of PVDF bound proteins for internal sequence analysis. Anal. Biochem. 218, 112–117.

    Article  PubMed  CAS  Google Scholar 

  29. Lui, M., Tempst, P., and Erdjument-Bromage, H. (1996) Methodical analysis of protein-nitrocellulose interactions to design a refined digestion protocol. Anal. Biochem. 241, 156–166.

    Article  PubMed  CAS  Google Scholar 

  30. Eckerskorn, C., and Lottspeich, F. (1989) Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix. Chromatographia 28, 92–94.

    Article  CAS  Google Scholar 

  31. Rosenfeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992) in-gel digestion of proteins for internal sequence analysis after one-or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173–179.

    Article  PubMed  CAS  Google Scholar 

  32. Jenö, P., Mini, T., Moes, S., Hintermann, E., and Horst., M. (1995) Internal sequences from proteins digested in polyacrylamide gels. Anal. Biochem. 224, 451–455.

    Article  Google Scholar 

  33. Hellman, U., Wernstedt, C., Gonez, J., and Heldin, C.-H. (1995) Improvement of in-gel digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224, 451–455.

    Article  PubMed  CAS  Google Scholar 

  34. 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.

    Article  PubMed  CAS  Google Scholar 

  35. Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., et al. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano electrospray mass spectrometry. Nature 379, 466–469.

    Article  PubMed  CAS  Google Scholar 

  36. Fernandez-Patron, C., Calero, M., Collazo, P. R., Garcia, J. R., Madrazo, J., Musacchio, A., et al. (1995) Protein reverse staining: high efficiency microanalysis of unmodified proteins detected on electrophoresis gels. Anal. Biochem. 224, 203–211.

    Article  PubMed  CAS  Google Scholar 

  37. Vorm, O., Roepstorff, P., and Mann, M. (1994) Matrix surfaces made by fast evaporation yield improved resolution and very high sensitivity in MALDI TOF. Anal. Chem. 66, 3281–3287.

    Article  CAS  Google Scholar 

  38. Vorm, O. and Mann, M. (1994) Improved mass accuracy in matrix assisted laser desorption/ionization time-of-flight mass spectrometry of peptides. J. Am. Soc. Mass Spectrom. 5, 955–958.

    Article  CAS  Google Scholar 

  39. Jensen, O. N., Podtelejnikov, A., and Mann, M. (1996) Delayed extraction improves specificity in database searches by MALDI peptide maps. Rapid Commun. Mass Spectrom. 10, 1371–1378.

    Article  PubMed  CAS  Google Scholar 

  40. Brown, R. S., and Lennon, J. J. (1995) Mass resolution improvement by incorporation of pulsed ion extraction/ionization linear time-of-flight mass spectrometry. Anal. Chem. 67, 1998–2003.

    Article  PubMed  CAS  Google Scholar 

  41. King, T. B., Colby, S. M., and Reilly, J. P. (1995) High resolution MALDI-TOF mass spectra of three proteins obtained using space-velocity correlation focussing. Int. J. Mass Spectrom. Ion Processes 145, L1–L7.

    Article  CAS  Google Scholar 

  42. Vestal, M. L., Juhasz, P., and Martin, S. A. (1995) Delayed extraction matrix-assisted laser desorption time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 9, 1044–1050.

    Article  CAS  Google Scholar 

  43. Whittal, R. M. and Li, L. (1995) High resolution matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer. Anal. Chem. 67, 1950–1954.

    Article  PubMed  CAS  Google Scholar 

  44. Jensen, O., Podtelejnikov, A., and Mann, M. (1997) Identification of the components of simple protein mixtures by high accuracy peptide mass mapping. Anal. Chem. 69(23), 4741–4750.

    Article  PubMed  CAS  Google Scholar 

  45. Henzel, W. J., Billeci, T. M., Stults, J. T., and Wong, S. C. (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. USA 90, 5011–5015.

    Article  PubMed  CAS  Google Scholar 

  46. Mann, M., HØjrup, P., and Roepstorff, P. (1993) Use of mass spectrometric molecular weight information to identify proteins in sequence databases. Biol. Mass Spectrom. 22, 338–345.

    Article  PubMed  CAS  Google Scholar 

  47. Pappin, D. J. C., HØjrup, P., and Bleasby, A. J. (1993) Protein identification by peptide mass fingerprinting. Curr. Biol. 3, 327–332.

    Article  PubMed  CAS  Google Scholar 

  48. James, P., Quadroni, M., Carafoli, E., and Gonnet, G. (1993) Protein identification by mass profile fingerprinting. Biophy. Biochem. Res. Commun. 195, 58–64.

    Article  CAS  Google Scholar 

  49. Yates, J. R., Speicher, S., Griffin, P. R., and Hunkapiller, T. (1993) Peptide mass maps: a highly informative approach to protein identification. Anal. Biochem. 214, 397–408.

    Article  PubMed  CAS  Google Scholar 

  50. Mann, M. (1994) Sequence database searching by mass spectrometric data, in Microcharacterization of Proteins (Kellner, R, Lottspeich, F., and Meyer, H. E., eds.), VCH Weinheim, pp. 223–245.

    Google Scholar 

  51. Jensen, O. N., Houthaeve, T., Shevchenko, A., Cudmore, S., Ashford, T., Mann, M., et al. (1996) Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis. J. Virol. 70, 485–7497.

    Google Scholar 

  52. Jensen, O. N., Vorm, O., and Mann, M. (1996) Sequence patterns produced by incomplete enzymatic digestion or one-step Edman degradation of peptide mixtures as probes for protein database searches. Electrophoresis 17, 938–944.

    Article  PubMed  CAS  Google Scholar 

  53. Jensen, O. N., Mortensen, P., Vorm, O., and Mann, M. (1997) Automation of MALDI mass spectrometry using fuzzy logic feedback control. Anal. Chem. 69, 1706–1714.

    Article  PubMed  CAS  Google Scholar 

  54. Houthaeve, T., Gausepohl, H., Mann, M., and Ashman, K. (1995) Automation of micro-preparation and enzymatic cleavage of gel electrophoretically separated proteins. FEBS Lett. 376, 91–94.

    Article  PubMed  CAS  Google Scholar 

  55. Ashman, K., Houthaeve, T., Clayton, J., Wilm, M., Podtelejnikov, A., Jensen, O. N., et al. (1997) The application of robotics and mass spectrometry to the characterization of the Drosophila melanogaster indirect flight muscle proteome. Lett. Pept. Sci. 4, 57–65.

    CAS  Google Scholar 

  56. Spengler, B., Kirsch, D., Kaufmann, R., and Jaeger, E. (1992) Peptide sequencing by matrix-assisted laser-desorption mass spectrometry. Rapid Commun. Mass Spectrom. 6, 105–108.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Protein Research Group, Department of Molecular Biology, Odense University, Odense, Denmark

    Ole N. Jensen

  2. Protein and Peptide Group, European Molecular Biology Laboratory, Heidelberg, Germany

    Matthias Wilm, Andrej Shevchenko & Matthias Mann

Authors
  1. Ole N. Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Matthias Wilm
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrej Shevchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthias Mann
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Molecular Biotechnology, University of Washington, Seattle, WA

    Andrew J. Link

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Jensen, O.N., Wilm, M., Shevchenko, A., Mann, M. (1999). Sample Preparation Methods for Mass Spectrometric Peptide Mapping Directly from 2-DE Gels. In: Link, A.J. (eds) 2-D Proteome Analysis Protocols. Methods in Molecular Biology, vol 112. Humana Press. https://doi.org/10.1385/1-59259-584-7:513

Download citation

  • DOI: https://doi.org/10.1385/1-59259-584-7:513

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-524-9

  • Online ISBN: 978-1-59259-584-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation