Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 2, 2000, pp. 119

Biotecnología Aplicada 2000;17:119

Goals and Feasible Techniques of Proteome Analysis
in the Year 2000

Brigitte Wittmann-Liebold,1 Christian Scheler2

1Max-Delbrueck-Centrum for Molecular Medicine, Robert-Roessle-Str. 10 D-13125 Berlin-Buch.
E-mail: liebold@mdc-berlin.de 2Wita GmbH. Warthestr. 21. 14513 Teltow,
Germany. E-mail: scheler@wita.de; http://www.wita.de

Code Number: ba00032

Proteome analysis offers several new approaches in bioscience research. Compared to genome analysis which defines a static situation of the cell, the proteome describes dynamic states of protein expression during cell development, differentiation or in cell disorders. Therefore characterization of protein extracts of different cell states becomes increasingly important for identification of disease-associated proteins, of development of early diagnosis markers, and for drug screening. Many proteins show variants and modifications which contribute to their functions like phosphorylation or glycosylation. In opposite, gene analysis does not allow to identify or predict these modifications and therefore only reflects a static situation of the inherited information. Furthermore, there is no direct correlation between mRNA and protein concentration, due to different half life times of mRNA and proteins and protein processing and translocation [1]. Nonetheless, the knowledge of genetic sequences stored in databases is essential for identification of proteins by mass spectrometric techniques used in proteome analysis.

A prerequisite for proteome analysis is a method for a high resolution separation of protein extracts of entire cells. At present two-dimensional electrophoresis (2DE) is the only available method of choice with a resolution of more than 5000 proteins. The best separation technique was developed by Klose and Kobalz [2], which enables these high resolutions in combination with unambiguous identification of the resulting protein spots. We used a combination of in-gel digestion [3] of the protein spots followed either by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) peptide mass fingerprinting or nano-electrospray ionization mass spectrometry (ESI-MS) for protein identification. We built up 2D-databases for heart muscle proteins, and for B-cell proteins identified at different stages in IgM-induced apoptosis.

Future techniques will implement more and more automation of the different analysis steps leading to high throughput characterization and identification of many proteins of the cell extracts. These possibilities open a wide range of new research fields, e.g. in antibiotic screening [4], identification of proteins associated with apoptosis [5], drug effects [6], infectious diseases [7] and cancer [8].

Such new techniques for proteome analysis, for instance, imply the development of automates of the 2DE method and reaction devices on wafer basis for the analysis and derivatization of minute sample amounts as developed in our lab. Further steps involve the combination and integration of the different partially automated wet laboratory techniques starting from two-dimensional separation, gel staining, spot cutting, in-gel digestion to mass spectrometry. Dry lab techniques like automatic spot detection, matching of 2DE gels, database searches and storage of information in suitable databases have also to be automated and combined with the wet lab methods to achieve a high throughput analysis of proteomics in the future.

References

1. Haynes PA, Gygi SP, Figeys D, Aebersold R. Proteome analysis: biological assay or data archive? Electrophoresis 1998;19: 1862–71.

2. Klose J, Kobalz U. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis 1995;1:1034-59.

3. Otto A. Thiede B, Müller EC, Scheler C, Jungblut, P. Identification of myocardial proteins by 2-D gel electrophoresis using an efficient sample preparation for mass spectrometry. Electrophoresis l996;17:1643–50.

4. Marzocchi B, Magi B, Bini L, Cellesi C, Rossolini A, Massidda 0, et al. Two-dimensional gel electrophoresis and immunoblotting of human serum albumin modified by reaction with penicillins. Electrophoresis 1995;16:851–3.

5. Brockstedt E, Rickers A, Bommert K, Dörken B, Wittmann-Liebold B, Otto A. Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. J Biol Chem 1998;273:28057–64

6. Anderson NL, Anderson NG. Proteome and proteomics: new technologies, new concepts. and new words. Electrophoresis 1998;19:1853–61

7. McAtee CP, Fry KE, Berg DE. Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by "proteome" technologies. Helicobacter 1998;3:163–9.

8. Zeindl-Eberhart E, Jungblut PR, Otto A, Rabes HM. ldentification of tumor-associated protein variants during rat hepatocarcinogenesis. Aldosereductase. J Biol Chem 1994;269:14589–94.

Papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.