Implementation of nano-liquid chromatography hyphenated to tandem mass spectrometry for protein identification in gel and non-gel based proteomics
This thesis is divided into three parts. Part I deals with the current status of large scale strategies for the analysis of proteins in biological systems. In Chapter I, genomics and the need to shift towards proteomic approaches are outlined. An overview of key technologies used in functional and structural proteomics is provided in Chapter II, whereas mass spectrometry and the strategies for profiling of proteins are discussed in detail in the final chapter of this part (Chapter III). Part II describes the analysis of cytosolic proteins by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. First, the techniques are highlighted that were used to identify gel separated proteins, i.e. the implementation of nano-liquid chromatography and mass spectrometry (Chapter IV). The next two chapters are applications of the developed methodology in two case studies: the protein composition of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 grown on ferric oxide (Chapter V) and the effect of a short-term heat shock on the plant barley (Chapter VI). Part III deals with the development of alternative strategies for the analysis of membrane proteins. A special electrophoretic technique, i.e. blue-native polyacrylamide gel electrophoresis, in combination with mass spectrometry, was used for profiling the different subunits of the oxidative phosphorylation system (Chapter VII), while multi-dimensional liquid chromatography coupled to MALDI tandem mass spectrometry was used for the profiling of the proteins present in the murine myelin sheath (Chapter VIII).
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