Development of Electrokinetically Based Two-Dimensional Separation Methodologies for Proteomic Studies

Proteomic studies require analytical methods capable of separating, identifying and quantifying thousands of proteins. Consequently, developing separation methodologies with high resolving power necessary to separate complex protein samples prior to protein identification via mass spectrometry (MS) has become a major challenge in proteomic studies. It is now demonstrated that a single separation technique is unable to provide a peak capacity sufficient to resolve complex protein mixtures, therefore multidimensional separation methodologies have to be developed. In this context, the highest peak capacity is obtained when the hyphenated methods present separation mechanisms as different as possible. Subsequently, the orthogonality of a two-dimensional (2-D) separation methodology has to be also evaluated as a feature that expresses the efficiency of hyphenation. The objectives of the thesis were mainly focused on the development and application of 2-D separation methodologies in proteomic studies. In the first part of this work, the potential of the combination of Capillary zone electrophoresis (CZE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is assessed. To further provide a bioanalytical platform with high-sensitivity capabilities, field-enhanced sample injection is integrated as on online preconcentration strategy upstream from the electrokinetic separation. Then, a novel approach to assess the orthogonality of 2-D separation systems based on conditional entropy is introduced. It considers the quantitative distribution of peaks in the entire separation space such that the orthogonality obtained is independent of the number of peaks observed for each separation technique. As another part of this work, the hyphenation of off-gel electrophoresis (OGE) with CZE is revised. While the application of the developed 2-D separation methodology is expanded to protein scale, a significant improvement of the practical peak capacity for peptide separation is also obtained. In this chapter, a computer based model is also developed which is able to digest a specific protein and simulate the 2-D OGE-CZE separation map of resulting peptides. Afterwards, a practical protocol for proteolysis assisted by trypsin loaded NH2-MOSF during OGE separation is developed. Besides savings in experimental effort and enhanced proteolysis efficiency, the straightforward assessment of pI values of peptides after simultaneous digestion and separation with OGE which facilitates peptide identification, is also one valuable aspect of the developed methodology. In the last part of the thesis, the proof of concept of the application of a push-pull microfluidic device for electrotransfer of proteins from a separation gel to a membrane is presented. The membrane can be then subjected to proteolysis and MALDI-MS analysis to visualize with high resolution the protein sample separated.


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