Development of cross-linking strategies for structural proteomics

Cameron Fyfe

Protein-protein interactions are critical to a vast array of biological processes and underpin mechanisms of virulence used by viral, bacterial and parasitic pathogens. Detailed data on protein-protein interactions are therefore critical to understanding of infection and developing intervention strategies to against pathogenic mechanisms. Mass spectrometry in conjunction with chemical cross-linking has the potential not only to enable us to identify interacting proteins, but can also yield detailed information on the site of interaction and the topology of protein complexes. However, despite improvements in instrumentation and the increased availability of a range of cross-linking chemistries there are still a number of challenges to overcome in order for these methods to become generally applicable. Solving these problems would revolutionise our ability to study protein-protein interactions on a cellular level.

We will use well characterised model protein-protein complexes that cover the range of affinities applicable to functional biological systems in order test the suitability of a range of cross-linking chemistries to study by mass spectrometry. Advanced instrumentation will allow us to apply multiple fragmentation methods to aid the ready identification of cross-linked peptides.  In addition to chemical cross-linking strategies we will also investigate the use of ‘protein cross-linking reagents’. These proteins, known as α-macroglobulins are activated by proteolytic cleavage of a ‘bait region’, and on activation covalently cross-link to the attacking protease via an activated thiol-ester group. Given the central role of proteases in pathogenesis, engineered affinity labelled α-macroglobulins with bait regions designed to capture proteases with a range of specificities would be an important tool in the identification of novel proteolytic virulence factors