Divide and conquer strategies for the in-depth analysis of proteomes

Global and in-depth protein characterisation is a key fundamental step in the unravelling of biological processes in all living organisms. However, the characterisation of the protein content (i.e. the proteome) of a cell, tissue or organism is an extremely challenging task due to the large number of proteins present, and their relative abundance spanning several orders of magnitude. Despite recent revolutionary developments in mass spectrometry (MS) based protein and peptide identification techniques, which have increased the speed and sensitivity for peptide sequencing, the reduction of the complexity of a proteome prior to MS analysis is currently still a prerequisite for in-depth proteome analysis. Therefore, numerous pre-fractionation techniques have been introduced for global proteomic profiling, which ideally result in an increase of the total number of protein identifications, evidently at the expense of an increase in analysis time. In this thesis, various protein and peptide pre-fractionation techniques have been explored, optimized and applied to the characterization of proteomes of several different origins ranging from cells to organisms, including Saccharomyces cerevisiae, Drosophila melanogaster and human Hek293 cells. These protein and peptide separation techniques have been used orthogonally or separately, but always in combination with high mass-accuracy mass spectrometers to not only identify the peptides, but additionally also to identify post-translational modifications, primarily phosphorylation and N-terminal acetylation. A significant part of this thesis has focused on the use of orthogonal separation techniques for the comprehensive characterisation of the yeast nuclear proteome. In this study, phosphocellulose p11 chromatography was evaluated as a generic pre-fractionation technique as well as an enrichment technique for DNA binding proteins. The work in this thesis has revealed that each of the used pre-fractionation techniques has its own merits. Therefore, the combined use of multiple approaches is of fundamental importance, especially for the identification of low abundant proteins as well as to increase the confidence of the identification of proteins and/or their post-translational modifications.