Subcellular protein fractionation in Legionella pneumophila and preparation of the derived sub-proteomes for analysis by mass spectrometry

Abstract

Classical proteomic techniques are perfectly suited to reflect changes in the metabolism by detection of changed protein synthesis rates and protein abundances in a global protein-centered analysis. Although the proteome of microbes is considered as rather low complex, usually the subcellular fractionation of proteins leads to higher proteome coverage which might be important for the proteome quantification. Additionally, such fractionation provides the possibility to detect changes in the protein localization as well as the protein abundance in single sub-proteomes. Here, a workflow for subcellular fractionation of Legionella pneumophila into cytosolic, periplasmic, membrane, and extracellular proteins for global proteome analyses is provided. The methods included in this workflow can be used to analyze the adaptation of L. pneumophila to different environmental and nutritional situations during infection or during different life cycle stages including planktonic or biofilm phase.