The aim of this PhD-Thesis was the development of a workflow, suitable for biomarker search within the proteome of blood cells.
Cellular proteins were separated by two-dimensional gel electrophoresis, quantified and those proteins, which showed different expression levels between the considered groups, were identified by matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. To achieve good and reproducible separation of the proteins by two-dimensional gel electrophoresis, an optimisation of the sample preparation steps was necessary. Therefore, the applicability of different precipitation agents and their compatibility to 2D electrophoresis was tested, whereas trichloroacetic acid has shown to be superior. To get an estimation of the expression changes, that have to be expected and to optimise protein identification workflow based on by mass spectrometry, an already established experimental setup for the analysis of the changes in the proteome of U937 cells (monocytic cell line), that were challenged by glutamine starvation, was used. The experiences that have been gained during the preliminary experiments were applied to the analysis of primary cells, thrombocytes. To design a search for biomarkers in thrombocytes, it is necessary to assess the amount of variability in protein expression levels between samples of different individuals. Therefore, the thrombocyte protein extracts, obtained from 20 volunteers, were analysed and the variation in protein expression levels within this group was determined. This degree of variation was used to estimate the minimum number of samples, that is needed to obtain statistical significance for a given difference in expression levels when comparing two groups of individuals. Some proteins showed very low variation and were identified by mass spectrometry. These proteins and can serve for normalisation purposes in different methods. Taking into account these results we are now able to define the technical procedure to perform a biomarker search in blood cells without further experiments.