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The importance of obtaining comprehensive and accurate information from cellular proteomics experiments asks for a systematic investigation of sample preparation protocols. In particular when working with unicellular organisms with strong cell walls, such as found in the model organism and cell factory Saccharomyces cerevisiae. Here, we performed a systematic comparison of sample preparation protocols using a matrix of different conditions commonly applied in whole cell lysate, bottom-up proteomics experiments. The different protocols were evaluated for their overall fraction of identified spectra, proteome and amino acid sequence coverage, GO-term distribution and number of peptide modifications, by employing a combination of database and unrestricted modification search approaches. Ultimately, the best protocols enabled the identification of approximately 65–70% of all acquired fragmentation spectra …