Development of functionalized cross-linking reagents

The study of biological pathways such as signaling pathways is being facilitated by continuous methodological and technological improvements. Today, proteomic technologies in combination with mass spectrometry provide the tools for an in-depth profiling of proteins in living systems. Techniques have become available that yield information beyond the mere cataloguing of proteins, e.g. on post-translational modifications and quantitative differences in protein expression. However, these methods focus on samples of solubilized, dissociated and denatured proteins. Since most biological processes are carried out not by individual proteins but rather by proteins that interact with each other in protein complexes most proteomic datasets lack the important contextual information. Protein complexes further interact with other complexes to form higher level interaction networks. While significant advances for the study of protein-protein interactions and protein complexes have been made recently, there is still an urgent need for further developments to allow the comprehensive analysis of protein complexes and interaction networks in living systems. Specifically, there is a need to determine the true composition of protein complexes in cells, the dynamic change in their composition in response to the changes in cellular state and to identify the topology of complexes, specifically the interfaces of their subunit components. Here, we propose the development of a new technology to study the topology of protein complexes on a system- wide level. It is based on a new class of functionalized cross-linking reagents and the mass spectrometric analysis of peptides cross-linked with these reagents. The cross-linkers will incorporate affinity tags to enable the selective isolation of labeled peptides out of complex peptide mixtures resulting from the digestion of cross-linked biosamples. Use of these optimized linkers will be combined with sophisticated mass spectrometric analysis and powerful bioinformatic tools that are unique to the chosen host laboratory and are expected to provide a new level of insight into protein complex organization. As a potential application field of this integrated chemical / mass spectrometric / bioinformatic method, we will focus on the interactions of cancer-related protein kinases and the profiling of the signal transduction pathways of this protein family.