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Structural Characterization of Proteins and Peptides

  • Rainer Deutzmann
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 94)

Abstract

The primary structure of proteins is nowadays determined by DNA sequencing, and a variety of genomes are already known. Nevertheless, protein sequencing/identification is still indispensable to analyze the proteins expressed in a cell, to identify specific proteins, and to determine posttranslational modifications. Proteins of interest are typically available in low microgram amounts or even less. The separation method of choice is gel electrophoresis, followed by blotting to PVDF membrane for N-terminal sequencing or by in-gel digestion to generate peptides that can be separated by HPLC. Structural analysis can be done by Edman degradation or mass spectrometry (MS). Edman degradation is the older method based on successive removal of N-terminal amino acids by chemical methods. Sequencing of a peptide requires many hours, the sensitivity is in the range of 2–5 pmol of a purified peptide. Nevertheless, Edman degradation is still the workhorse in the lab for routine work such as identification of blotted proteins. It is also the method of choice for sequencing unknown proteins/peptides and modified peptides. MS has routinely been used with peptides in the range of 100 fmol or even less. In contrast to Edman degradation, complex mixtures such as tryptic digests can be analyzed, making HPLC separation of peptides unnecessary. MS is a very fast method that can be automated. It is the method of choice for sensitive analysis and large-scale applications (proteomics). Two different ionization methods are commonly used to generate peptide/protein ions for MS analysis. These are MALDI (matrix assisted laser desorption and ionization) and ESI (electrospray ion-ization). They can be combined with a variety of mass analyzers (TOF, quadrupole, ion trap). Proteins are either identified by searching databases with the masses of proteolytic peptides (peptide mass fingerprinting) or using fragmentation data (raw MS/MS spectra or sequence tags). This approach requires that the protein is known and listed in the database. De novo sequencing by MS of peptides is possible, but very time consuming and not a routine application, in contrast to Edman degradation. The aim of this chapter is to introduce to basic theory, practical applications and limitations of the various methods, to enable the non-expert scientist to decide which method is best suited for his project and which kind of sample preparation is necessary.

Keywords

Sodium Dodecyl Sulfate Edman Degradation Pyroglutamic Acid Weak Organic Acid Proteolytic Peptide 
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.

References

  1. 1.
    Walker, J. M., ed. (2002) The Protein Protocols Handbook. Humana, Totowa, NJ.Google Scholar
  2. 2.
    Howard, G. C. and Brown, W. E., eds. (2002) Modern Protein Chemistry: Practical Aspects. CRC Press, Boca Raton.Google Scholar
  3. 3.
    Smith, B. J., ed. (1997) Protein sequencing protocols, in Methods in Molecular Biology, vol. 64 (Walker, J. M., ed.), Humana, Totowa, NJ.Google Scholar
  4. 4.
    Kellner, R., Lottspeich, F., and Meyer, H. E., eds. (1994) Microcharacterization of Proteins. VCH, Weinheim.Google Scholar
  5. 5.
    Matsudaira, P., ed. (1993) A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic, San Diego.Google Scholar
  6. 6.
    Allen, G. (1989) Sequencing of proteins and peptides, in Laboratory Techniques in Biochemistry and Molecular Biology, vol. 9 (Burdon, R. H. and van Knippenberg, P. H., eds.), Elsevier, Amsterdam.Google Scholar
  7. 7.
    Darbre, A. (1986) Practical Protein Chemistry—A Handbook, John Wiley & Sons, New York.Google Scholar
  8. 8.
    Gooding, K. M. and Regnier, F. E., eds. (2002) HPLC of Biological Macromolecules, 2nd ed., in Chromatographic Science Series, vol. 87, Marcel Dekker, New York.Google Scholar
  9. 9.
    Oliver, R. W. A., ed. (1998) HPLC of Macromolecules, in The Practical Approach Series (Rickwood, D. and Hames, B. D., eds.), IRL, Oxford.Google Scholar
  10. 10.
    Podell, D. N. and Abraham, G. N. (1978) A technique for the removal of pyroglutamic acid from the amino terminus of proteins using calf liver pyrogutamate amino peptidase. Biochem. Biophys. Res. Commun. 81, 176–185.PubMedCrossRefGoogle Scholar
  11. 11.
    Hirano, H., Komatsu, S., and Tsunagawa, S. (1997) On-membrane deblocking of proteins, in Protein Sequencing Protocols (Smith, B. J., ed.), Humana, Totowa, NJ, pp. 285–292.Google Scholar
  12. 12.
    Bergmann, T., Gheorghe, M. T., Hjelmqvist, L., and Jörnvall, H. (1996) Alcoholytic deblocking of N-terminally acetylated peptides and proteins for sequence analysis. FEBS Lett. 390, 199–202, and references therein.CrossRefGoogle Scholar
  13. 13.
    Gravel, P. (2002) Protein blotting by the semidry method, in The Protein Protocols Handbook (Walker, J. M., ed.), Humana, Totowa, NJ, pp. 321–334.CrossRefGoogle Scholar
  14. 14.
    Eckerskorn, C. (1994) Electroblotting, in Microcharacterization of Proteins (Kellner, R., Lottspeich, F., and Meyer, H. E., eds.), VCH, Weinheim, pp. 75–92.CrossRefGoogle Scholar
  15. 15.
    Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262, 10,035–10,038.PubMedGoogle Scholar
  16. 16.
    Eckerskorn, C., Mewes, W., Goretzki, H. W., and Lottspeich, F. (1988) A new siliconized-glass fiber as a support for protein chemical analysis of electroblotted proteins. Eur. J. Biochem. 176, 509–519.PubMedCrossRefGoogle Scholar
  17. 17.
    Bjerrum, O. J. and Schafer-Nielson, C. (1986) Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus, in Electrophoresis 1986 (Dunn, M. J., ed.), VCH, Weinheim, pp. 315–327.Google Scholar
  18. 18.
    Towbin, H., Staehlin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.PubMedCrossRefGoogle Scholar
  19. 19.
    Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E., and Kent, S. B. (1987) Internal amino acid sequence analysis of proteins separated by one-or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. USA 84, 6970–6974.PubMedCrossRefGoogle Scholar
  20. 20.
    Fernandez, J. and Mische, S. M. (2002) Enzymatic digestion of membrane-bound proteins for peptide mapping and internal sequence analysis, in The Protein Protocols Handbook (Walker J. M., ed.), Humana, Totowa, NJ, pp. 523–532.CrossRefGoogle Scholar
  21. 21.
    Szewczyk, B. and Summers, D. (1988) Preparative elution of proteins blotted to immobilon membranes. Anal. Biochem. 168, 48–53.PubMedCrossRefGoogle Scholar
  22. 22.
    Jenö, P. and Horst, M. (2002) Electroelution of proteins from polyacrylamide gels, in The Protein Protocols Handbook (Walker, J. M., ed.), Humana, Totowa, NJ, pp. 299–305.CrossRefGoogle Scholar
  23. 23.
    Amons, R. (1987) Vapor-phase modification of sulfhydryl groups in proteins. FEBS Lett. 212, 68–72.PubMedCrossRefGoogle Scholar
  24. 24.
    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
  25. 25.
    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.PubMedCrossRefGoogle Scholar
  26. 26.
    Aguilar, M.-I. and Hearn, M. (1996) High resolution reverse-phase high-performance liquid chromatography of peptides and proteins. Methods Enzymol. 270, 3–26.PubMedCrossRefGoogle Scholar
  27. 27.
    Stone, K. L. and Williams, K. R. (2002) Reverse-phase HPLC separation of enzymatic digests of proteins, in The Protein Protocols Handbook (Walker, J. M., ed.), Humana, Totowa, NJ, pp. 533–540.CrossRefGoogle Scholar
  28. 28.
    Lottspeich, F., Houthave, T., and Kellner, R. (1994) The Edman degradation, in Microcharacterization of Proteins (Kellner, R., Lottspeich, F., and Meyer, H. E., eds.), VCH, Weinheim, pp. 117–130.CrossRefGoogle Scholar
  29. 29.
    Hunkapiller, M. W., Hewick, R. M., Dreyer, W. J., and Hood, L. E. (1983) High-sensitivity sequencing with a gas-phase sequenator. Methods Enzymol. 91, 399–413.PubMedCrossRefGoogle Scholar
  30. 30.
    Chapman, J. R., ed. (1996) Protein and peptide analysis by mass spectrometry, in Methods in Molecular Biology (Walker, J. M., ed.), Humana, Totowa, NJ.Google Scholar
  31. 31.
    Dass, C. (2001) Principles and Practice of Biological Mass Spectrometry, John Wiley & Sons, New York.Google Scholar
  32. 32.
    Karas, M. and Hillenkamp, F. (1988) Laser desorption ionisation of proteins with molecular masses exceeding 10000 D. Anal. Chem. 60, 2299–2301.PubMedCrossRefGoogle Scholar
  33. 33.
    Beavis, J. F. and Chait, B. T. (1996) Matrix-assisted laser desorption ionization mass-spectrometry of proteins. Methods Enzymol. 270, 519–551.PubMedCrossRefGoogle Scholar
  34. 34.
    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.CrossRefGoogle Scholar
  35. 35.
    Brown, R. S. and Lenon, J. J., (1995) Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometer. Anal. Chem. 67, 1998–2002.PubMedCrossRefGoogle Scholar
  36. 36.
    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.PubMedCrossRefGoogle Scholar
  37. 37.
    Banks, J. F. and Whitehouse, C. M. (1996) Electrospray ionization mass spectrometry. Methods Enzymol. 270, 486–518.PubMedCrossRefGoogle Scholar
  38. 38.
    Wilm, M. and Mann, M. (1996) Analytical properties of the nano electrospray ion source. Anal. Chem. 66, 1–8.CrossRefGoogle Scholar
  39. 39.
    Jensen, O. N. and Wilm, M. (2002) Peptide sequencing by nanospray tandem mass spectrometry. in The Protein Protocols Handbook (Walker, J. M., ed.), Humana, Totowa, NJ, pp. 693–710.CrossRefGoogle Scholar
  40. 40.
    Dawson, P. H. (1995) Quadrupole Mass Spectrometry and Its Applications, AIP Press, Woodbury, NY.Google Scholar
  41. 41.
    Jonscher, K. R. and Yates III, J. R. (1997) The quadrupole ion trap mass spectrometer—A small solution to a big challenge. Anal. Biochem. 244, 1–15.PubMedCrossRefGoogle Scholar
  42. 42.
    Shevchenko, A., Chernushevich, I., Wilm, M., and Mann, M. (2000) De novo peptide sequencing by nanoelectrospray tandem mass spectrometry using triple quadrupole and quadrupole/time-of-flight instruments, in Mass Spectrometry of Proteins and Peptides (Chapman, J. R., ed.), Humana, Totowa, NJ, pp. 1–16.CrossRefGoogle Scholar
  43. 43.
    Lamond, A. I. and Mann, M. (1997) Cell biology and the genome projects—a concerted strategy for characterizing multiprotein complexes by using mass spectrometry. Trends Cell Biol. 7, 139–142.PubMedCrossRefGoogle Scholar
  44. 44.
    Patterson, S. D. (1997) Identification of low to subpicomolar quantities of electrophoretically separated proteins: towards protein chemistry in the post-genome era. Biochem. Soc. Trans. 25, 255–262.PubMedGoogle Scholar
  45. 45.
    Fenyö, D. (2000) Identifying the proteome: software tools. Curr. Opin. Biotechnol. 11, 391–395.PubMedCrossRefGoogle Scholar
  46. 46.
    Roepstorff, P. and Fohlmann, J. (1984) Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed. Mass Spectrom. 11, 601–611.PubMedCrossRefGoogle Scholar
  47. 47.
    Martin, S. E., Shabanowitz, J., Hunt, D. F., and Marto, J. A. (2000) Subfemtomol MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 72, 4266–4276.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Rainer Deutzmann
    • 1
  1. 1.Institute for BiochemistryUniversity of RegensburgRegensburgGermany

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