High-resolution 2DE.

About 30 years ago two-dimensional gel electrophoresis (2DE) was developed independently by Klose and O'Farrell representing the combination of two orthogonal separation techniques. In the first dimension the proteins are separated by isoelectric focusing (IEF) according to their isoelectric point. In the second dimension proteins are separated according to their electrophoretic mobility by conventional SDS-PAGE. For IEF two different techniques, immobilized pH gradient (IPG) and carrier-ampholyte-based IEF (CA-based IEF), respectively, are currently applied. With a resolution of up to 10,000 protein spots in one gel, 2DE offers a huge potential to give a comprehensive overview of the proteins present in the examined system. In combination with image analysis and mass spectrometry 2DE is still the method of choice to analyse complex protein samples.In this chapter we provide detailed protocols for both 2DE systems and give an overview about the latest developments including the two-dimensional difference gel electrophoresis (DIGE) system.

Pilot study of the Human Proteome Organisation Brain Proteome Project: applying different 2-DE techniques to monitor proteomic changes during murine brain development.

The Human Proteome Organisation Brain Proteome Project aims at coordinating neuroproteomic activities with respect to analysis of development, aging, and evolution in human and mice and at analysing normal aging processes as well as neurodegenerative diseases. Our group participated in the mouse pilot study of this project using two different 2-DE systems, to find out the optimal conditions for comprehensive gel-based differential proteome analysis. Besides the assessment of the best methodical conditions the question of "How many biological replicate analyses have to be performed to get reliable statistically validated results?" was addressed. In total 420 differences were detected in all analyses. Both 2-DE methods were found to be suitable for comprehensive differential proteome analysis. Nevertheless, each of the methods showed substantial advantages and disadvantages resulting in the fact that modification of both systems is essential. From our results we can draw the conclusions that for the future optimal quantitative differential gel-based brain proteome analyses the sample preparation has to be slightly changed, the resolution of the first as well as the second dimension has to be advanced, the number of experiments has to be increased and that the 2D-DIGE system should be applied.

2-D differential membrane proteome analysis of scarce protein samples.

Proteome studies with small sample amounts are difficult to perform, especially when membrane proteins are the focus of interest. In our study a new method for the analysis of scarce membrane protein samples combining large gel 2-D-CTAB/SDS-PAGE with fluorescence dye saturation labelling (satDIGE) was developed, allowing a highly sensitive differential analysis of different cell states. After Triton X-114 phase partitioning, enriched membrane protein samples of T cells were labelled at cysteine residues using fluorescence dyes and separated by large gel 2D-CTAB/SDS-PAGE. For a differential analysis 3 mug protein was found to be sufficient to detect proteins in a widespread well-separated diagonal spot pattern.

A peptide preconcentration approach for nano-high-performance liquid chromatography to diminish memory effects.

An advanced method has been developed for the analysis of proteolytic digests of complex protein mixtures by reversed phase high-performance liquid chromatography coupled to mass spectrometry. The occurrence of memory effects was prevented by a parallel set of two precolumns employed for simultaneous separation and washing procedures. The system was tested extensively, and tryptic digests of three single proteins were analyzed. In addition, different solvent systems were evaluated for effective washing of the employed precolumns. Using the analytical strategy presented, a reliable identification of proteins in complex mixtures was obtained and not hampered by the occurrence of memory effects.

The proteome of Saccharomyces cerevisiae mitochondria.

We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent approximately 90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.