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Results from biochemical studies have clearly indicated that most biological functions are not carried out by single polypeptides, but rather by macromolecular complexes, some of which dynamically assemble or disassemble as a function of cellular state. Results from systems biology studies have indicated that such macromolecular complexes are further ordered in extensive networks of interacting molecules and that important properties of such networks are the direct consequence of network structure. In every cell, at any given time, numerous types of molecular networks are concurrently present. Among these protein:protein interaction networks are of particular interest because they contain the modules that directly control and catalyze most cellular processes.
We have developed a robust platform to study the composition, structure and dynamics of protein:protein complexes and protein:protein interaction networks that consists of wet laboratory and computational modules. In this presentation we will discuss three specific modules of the technology and demonstrate their performance with selected applications. First, we will describe an optimized method for the generation of human cell lines expressing affinity tagged proteins and optimized protocols for AP-MS studies which now allow us to routinely determine protein complex compositions from 3x 10exp cells. Second, we will describe a method for the quantitative analysis of protein complexes that allows us to determine complex component stoichiometry and to detect changes in protein complex composition. Third, we will describe the use of isotope tagged cross linking reagents and mass spectrometry to generate distance constraints for the computational modeling of complex topology and structure. Collectively, these methods constitute important steps towards the analysis of the cellular interaction proteome in space and time. |
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