Z01: Characterization of membrane protein complexes by protein mass spectrometry
The central project Z01 provides the infrastructure and know-how for state-of-the-art mass spectrometry allowing to characterize membrane proteomes and membrane protein complexes. For the understanding of the organization of the cell - the basic biological unit of all known living organisms - membrane proteins play a pivotal role. Approximately 30% of the protein-coding genes express membrane proteins. However due to their physicochemical properties and their low copy numbers in contrast to water soluble proteins there is a lack of detailed knowledge how membrane proteins achieve their biological function and contribute to cellular structures, function and compartmentalization. In the last years mass spectrometry (MS) in combination with sophisticated sample preparation and separation techniques has proven to be an indispensable tool to characterize membrane proteins and their complexes. Z01 will take advantage of the MS-based proteomic workflow already established at the Molecular Proteomics Laboratory (MPL) for the identification of interaction partners and posttranslational modifications using affinity purification mass spectrometry (AP-MS) in combination with protein quantification and high-resolution mass spectrometry, respectively. Therefore, Z01 provides access to modern mass spectrometers (two Q Exactive plus systems, Orbitrap Fusion Lumos and TSQ Vantage) for qualitative and quantitative protein analysis. Quantitative approaches become possible through isotopic labeling and label-free strategies. Such strategies permit the quantitative analysis of complex composition. Further, through combination of mass spectrometry with various affinity purification methods (pull-down assays, immunoprecipitation etc.) and cross-linking experiments, highly accurate and sensitive analyses of protein complex compositions will become feasible. These methods will complement the in vivo imaging and NMR spectroscopic approaches undertaken in several subprojects. As there is great demand in the in detailed characterization of membrane protein complexes Z01 will in close collaboration with the project partners apply protein crosslinking in living cells followed by high-resolution mass spectrometry (XL-MS). Therewith Z01 will contribute to the understanding of stable and transient protein complex formation in time and space. To understand the underlying molecular principles of interaction and regulation of membrane proteins, we will start with a broad perspective including classic studies of isolated systems under defined conditions up to systems in the in vivo environment that undergo dynamic changes in interaction and composition. Protein mass spectrometry is one cornerstone to decipher these processes because only the combined knowledge of structure, function, and dynamics will lead to a complete picture of membrane protein complexes. And only such a complete picture will help to unravel how membrane protein complexes and their static and/or dynamic changes establish a complex biological network that spans a biological membrane and regulates cellular responses to internal and external stimuli.
Project leader: Prof. Dr. Kai Stühler, ,
Researchers: Dr. Nina Prescher (parental leave),