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Mass spectrometry-based cellular proteomics has taken a prominent role in many fields of research, including life sciences, biotechnology and microbial ecology. Still, advanced mass spectrometry-based proteomics methods are not routinely applied to microbes. The cell factory and model organism Saccharomyces cerevisiae, for example, is very well characterized, however, many research questions surrounding proteome dynamics under different growth conditions, and the regulation of its complex metabolic network via post-translational modifications, remain to be answered. Therefore, the aim of this thesis was to establish and apply optimized protocols to enable the large-scale quantitative analysis of yeast proteome dynamics under highly controlled conditions. In addition, a novel mass spectrometric approach was established to quantify the degree of modification of metabolic enzymes, that allows for a better understanding of their role in metabolic regulation.