Summary
The rapid development in proteomics over the last 10 years has led to a series of new technologies and combinations of them designed to unravel as much as possible of the proteins of an organism or otherwise specified biological material. Despite being a little tricky at certain steps, 2-DE has a very high resolution power with more than 10,000 spots per gel and is able to separate one protein into its different protein species caused by posttranslational modifications, alternative splicing or genetic variability. This high-resolution separation is combined with a highly sensitive identification method using peptide mass fingerprinting combined with sequence information by MS/MS, which results in high sequence coverage: the key to elucidate protein species structures. The off-line measurement by MALDI-TOFTOF-MS allows the repeated measurement of each sample and therefore provides more complete structure information for each protein species. The presented protocols represent the basic technology consisting of 2-DE, two staining methods, tryptic digestion and MALDI-TOFTOF-MS.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Macko, V. and Stegemann, H. (1969) Mapping of potato proteins by combined electrofocusing and electrophoresis identification of varieties. Hoppe-Seyler's Z. Physiol. Chem. 350, 917–919.
Kaltschmidt, E. and Wittmann, H.G. (1970)Ribosomal proteins. VII Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Anal. Biochem. 36, 401–412.
Vesterberg, O. and Svensson, H. (1966)Isoelectric fractionation, analysis and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with the separation of myoglobins. Acta Chem. Scand. 20, 820–834.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
O'Farrell, P.H. (1975) High-resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Klose, J. and Kobalz, U. (1995) Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electro-phoresis 16, 1034–1059.
Westermeier, R., Postel, W., Weser, J., and oerg, A. (1983) High-resolution 2-DE with IEF in immobilized pH gradients. J. Biochem. Biophys. Methods 8, 321–330.
Towbin, H., Staehelin, T., and Gordon, J.(1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. U. S. A. 76, 4350–4354.
Vandekerckhove, J., Bauw, G., Puype, M.,Van Damme, J., and Van Montagu, M. (1985) Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/poly-acrylamide gel. Eur. J. Biochem. 152, 9–19.
Eckerskorn, C., Jungblut, P., Mewes, W., Klose, J., and Lottspeich, F. (1988) Identification of mouse brain proteins after two-dimensional electrophoresis and elec-troblotting by microsequence analysis and amino acid composition analysis. Electro-phoresis 9, 830–838.
Karas, M. and Hillenkamp, F. (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 60, 2299–2301.
Fenn, J.B., Mann, M., Meng, C.K., Wong,S.F., and Whitehouse, C.M. (1989) Electro-spray ionization for mass spectrometry of large biomolecules. Science 246 (4926), 64–71.
Pappin, D.J., Hojrup, P., and Bleasby, A.J.(1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol. 3, 327–332.
Medzihradszky, K.F., Campbell, J.M., Bald-win, M.A., Falick, A.M., Juhasz, P., Vestal, M.L., and Burlingame, A.L. (2000) The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer. Anal. Chem. 72, 552–558.
Klose, J. (1999) Large-gel 2-D electro-phoresis, In Methods in Molecular Biology 112: 2-D Proteome Analysis Protocols (Link, A.J., (ed.), Humana Press, Totowa, NJ, pp 147–172.
Rabilloud, T. (1999) Solubilization of proteins in 2-D electrophoresis, In Methods in Molecular Biology 112: 2-D Proteome Analysis Protocols (Link, A.J., (ed.), Humana Press, Totowa, NJ, pp 9–19.
Jungblut, P.R., and Seifert, R. (1990) Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells. J. Biochem. Biophys. Methods 21, 47–58.
Doherty, N.S., Littman, B.H., Reilly, K.,Swindell, A.C., Buss, J.M., and Anderson, N.L. (1998) Analysis of changes in acute-phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis. Electrophoresis 19, 355–363.
Scheler, C., Lamer, S., Pan, Z., Li, X.P., Salnikow, J., and Jungblut, P. (1998) Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensionalelectrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrom-etry (MALDI-MS). Electrophoresis 19, 918–927.
Miller, I., Crawford, J., and Gianazza, E.(2006) Protein stains for proteomic applications: which, when, why? Proteomics 6, 5385–5408.
Jungblut, P.R., Bumann, D., Haas, G.,Zimny-Arndt, U., Holland, P., Lamer, S., Siejak, F., Aebischer, A., and Meyer, T.F. (2000) Comparative proteome analysis of Helicobacter pylori. Mol. Microbiol. 36, 710–725.
Schmidt, F., Donahoe, S., Hagens, K., Mattow, J., Schaible, U.E., Kaufmann, S.H., Aebersold, R., and Jungblut, P.R. (2004) Complementary analysis of the Mycobacte-rium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology. Mol. Cell. Proteomics 3, 24–42.
Schmidt, F., Schmid, M., Facius, A., Mattow, J., Pleissner, K.-P., and Jungblut, P.R. (2003) Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis. JASMS 14, 943–956.
Schmidt, F., Krah, A., Schmid, M., Jungblut, P.R., and Thiede, B. (2006) Distinctive mass losses of tryptic peptides generated by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight. Rapid Com-mun. Mass Spectrom. 20, 933–936.
Hoehenwarter, W., Ackermann, R., Zimny- Arndt, U., Kumar, N.M., and Jungblut, P.R. (2006) The necessity of functional pro-teomics: protein species and molecular function elucidation exemplified by in vivo alpha A crystallin N-terminal truncation. Amino Acids 31, 317–323.
Okkels, L.M., Müller, E.C., Schmid, M., Rosenkrands, I., Kaufmann, S.H., Andersen, P., Jungblut, P.R. (2004) CFP10 discriminates between nonacetylated and acetylated ESAT-6 of Mycobacterium tuberculosis by differential interaction. Proteomics 4, 2954–2960.
Acknowledgements
We thank Prof. Klose from The Institute of Human Genetics, Charité Berlin, who helped with developing the basis of the 2-DE technology presented here and coworkers from MPIIB for many useful discussions.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zimny-Arndt, U., Schmid, M., Ackermann, R., Jungblut, P.R. (2009). Classical Proteomics: Two-Dimensional Electrophoresis/MALDI Mass Spectrometry. In: Lipton, M.S., Paša-Tolic, L. (eds) Mass Spectrometry of Proteins and Peptides. Methods In Molecular Biology, vol 492. Humana Press. https://doi.org/10.1007/978-1-59745-493-3_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-493-3_4
Publisher Name: Humana Press
Print ISBN: 978-1-934115-48-0
Online ISBN: 978-1-59745-493-3
eBook Packages: Springer Protocols