Heart High-Performance 2-DE Database

History

The idea of 2-DE databases arose with the first description of the protein separation from E. coli (O'Farrell, 1975) and mouse tissues (Klose, 1975) by the combination of isoelectric focusing and SDS-polyacrylamide gel electrophoresis. The human protein index was proposed by Anderson in 1981 (Clark, 1981). In the laboratory of Prof. Klose we worked very hard on the idea of a systematic analysis of mouse brain and liver proteins (Jungblut and Klose, 1985, 1986, 1989, Klose, 1989). We soon recognized that small gels are not sufficient in resolution to resolve a substantial amount of all proteins of a tissue. To improve resolution two strategies were tried, prefractionation by chromatography or by cell fractionation and increasing the gel size. Both strategies showed that the number of proteins in the investigated mouse tissues is clearly higher than 10 000. A tissue as homogeneous as possible, for example heart tissue, should contain lower amounts of different proteins. The second most important prerequisite for the construction of 2-DE databases was the identification of proteins from 2-DE gels. Aebersold et al., 1986 and Vandekerckhove et al., 1986 developed membranes from which proteins after blotting from gels could be sequenced. I learned these techniques at the Max Planck Institute in Martinsried from Dr. Lottspeich and Dr. Eckerskorn. Identification of proteins from our large 2-DE gels by N-terminal sequencing and amino acid analysis was first published in this cooperation (Eckerskorn et al., 1988).

With these prerequisites, high resolution and identification, I started the construction of a human myocardial database in 1990 still in the laboratory of Prof. Klose. In 1991 I was engaged at the German Heart Center in Berlin, where I built up the 2-DE laboratory and tried to identify in cooperation with Dr. Otto from Max Delbrück Center the first proteins of our 2-DE patterns (Jungblut et al., 1992a). This database was extended with identification data (Jungblut et al., 1994). At that time two problems limited the progress: The 2-DE system, was not commercially available and the sensitivity of identification was not sufficient to identify more than the most intensive spots of a gel. Therefore, Prof. Wittmann-Liebold and I founded a company, Wittmann Institute of Technology and Analysis of Biomolecules (WITA), in 1992. WITA sells equipment, service and know-how of the 2-DE technique developed by Klose. The method is exhaustively described with each detail, also for a further increase in gel size up to 30 x 40 cm, by Klose and Kobalz, 1995.

The sensitivity of identification was improved during the next years in the collaboration of WITA with the Protein chemistry group at the Max Delbrück Center. Whereas in the first years N-terminal sequencing and amino acid analysis were the most preferred methods for identification (Jungblut et al., 1992b), today internal sequencing and mass spectrometry are more effective (Thiede et al., Jungblut et al., Otto et al., Müller et al., 1996). We found that our data are in relatively good accordance with the data of Harefield heart database. After a careful check Dr. Müller created the heart high-performance 2-DE database update 1996. I hope this database will be a help for the project of protein analysis on the genomic level (Jungblut and Wittmann-Liebold, 1995) or proteome analysis (Kahn, 1995).

Nov 22nd, 1996, P. Jungblut

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Since March 2007 the database is hosted at the Max Planck Institute for Infection Biology.

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References

Aebersold, R.H., Teplow, D.B., Hood, L.E., and Kent, S.B. (1986) Electroblotting onto activated glass: High efficiency preparation of proteins from analytical sodium dodecyl sulfate polyacrylamide gels for direct sequence analysis. J.Biol.Chem. 261, 4229-4238.

Clark, B.F.C. (1981) Towards a total human protein map. Nature 292, 491-492.

Eckerskorn, C., Jungblut, P., Mewes, W., and Klose, J. Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis. Electrophoresis 9, 830-838.

Jungblut, P. and Klose, J. (1985) Genetic variability of proteins from mitochondria and mitochondrial fractions of mouse organs. Biochem. Genet. 23, 227-245.

Jungblut, P. and Klose, J. (1986) Composition and genetic variability of Heparin-Sepharose CL-6B protein fractions obtained from the solubilized proteins of mouse organs. Biochem. Genet. 24, 925-939.

Jungblut, P., and Klose, J. (1989) Dye ligand chromatography and two-dimensional electrophoresis of complex protein extracts from mouse tissue. J. Chromatogr. 482, 125-132.

Jungblut, P., Otto, A., Regitz, V., Fleck, E., and Wittmann-Liebold, B., (1992a), Identification of human myocardial proteins separated by two-dimensional electrophoresis. Electrophoresis 13, 739-741.

Jungblut, P., Dzionara, M., Klose, J., and Wittmann-Liebold, B. (1992b) Identification of tissue proteins by amino acid analysis after purification by two-dimensional electrophoresis. J.Prot.Chem. 11, 603-612.

Jungblut, P., Otto, A., Zeindl-Eberhart, E., Pleissner, K.-P., Knecht, M., Regitz-Zagrosek, V., Fleck, E., and Wittmann-Liebold, B., (1994) Protein composition of the human heart: The construction of a myocardial two-dimensional electrophoresis database, Electrophoresis 15, 685-707.

Jungblut, P., and Wittmann-Liebold, B., (1995) Protein analysis on a genomic scale. J.Biotechnol. 41, 111-120.

Jungblut, P., Thiede, B., Zimny-Arndt, U., Müller, E.-C., Scheler, C., Wittmann-Liebold, B., and ,Otto, A., (1996) Resolution power of two-dimensional electrophoresis and identification of proteins from gels. Electrophoresis 17, 839-847.

Kahn, P., (1995) From genome to proteome. Science 270, 369-370.

Klose, J., (1975) Protein mapping by combined isoelectric focusing and electrophoresis in mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26, 231-243.

Klose, J. (1989) Systematic analysis of the total proteins of a mammalian organism: Principles and problems; Implications for sequencing the human genome. Electrophoresis 10, 140-152.

Klose, J. and Kobalz, U. (1995) Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis 16, 1034-1059.

Müller, E.-C., Thiede, B., Zimny-Arndt, U., Scheler, C., Prehm, J., Müller-Werdan, U., Wittmann-Liebold, B., Otto, A., and Jungblut, P. , (1996) High-performance human myocardial two-dimensional electrophoresis database: Edition 1996. Electrophoresis 17, in press.

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

Otto, A., Thiede, B., Müller, E.-C., Scheler, C., Wittmann-Liebold, B., and Jungblut, P. (1996) Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry, Electrophoresis 17, 1643-1650.

Thiede, B., Otto, A., Zimny-Arndt, U., Müller, E.-C., and Jungblut, P., (1996) Identification of human myocardial proteins separated by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 17, 588-599.

Vandekerckhove, J., Bauw, G., Puype, M., Van Damme, J., and Van Montagu, M. (1985) Protein-blotting on polybrene-coated glass-fiber sheets. A basis of acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on SDS-polyacrylamide gel. Eur.J.Biochem. 152, 9-19.

Acknowledgements

The project was financed by Deutsche Forschungsgemeinschaft (Forschungsprojekt Kl 237/3-1, Systematische Analyse der Zellproteine) and for the characterization of stress proteins (Forschungsprojekt Sa 267/10-1 Proteinchemische Charakterisierung von bei Herzinsuffizienz auftretenden Isoformen des Streßproteins Hsp27 im Myokard).

Further we thank for financial support obtained by WITA.

We thank for contributions, help and fruitful discussions

We acknowledge especially B. Thiede for introducing MALDI-MS identifications, B. Wittmann-Liebold for protein sequencing know-how, financial and organizational support, and J. Klose for 2-DE know-how and many discussions leading to the idea to construct a human 2-DE myocardial database.