Photocleavable crosslinker design, synthesis and analysis for enhanced crosslinking mass spectrometry methods

• Applicant: Professor Dr. Juri Rappsilber
• Subject Area: Analytical Chemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 449713269

Crosslinking mass spectrometry is a multi-scale, protein behaviour determination technology. It is now an established and robust tool for elucidating structures of individual proteins and complexes. The method is currently under active development for use in the study of protein conformational changes and protein-protein interactions. Crucially, crosslinking mass spectrometry has the capacity to achieve all of the above, in the native environment in which proteins exist, the living cell. The method can also scale further, to systems level and proteome-wide analysis, on cell lysates, organelles and intact cells. However, higher levels of protein mixture complexity create new challenges for analysis.Crosslinking mass spectrometry technology is underpinned by the chemistry used to introduce covalent links, which preserve the critical spatial information. Crosslinker development is therefore a key driver of crosslinking mass spectrometry method development. Novel crosslinkers can enhance the analytical process with their interplay between data acquisition and data analysis approaches. Recent progress in ultraviolet photodissociation (UVPD) opens a new angle here, which we now want to exploit for crosslinking mass spectrometry. We will develop chemical crosslinkers with enhanced functionality (reactivity and crosslinker UV-cleavability), as well as new analytical and computational methods for better utilisation of the resulting data. We expect to achieve significantly greater depth for high-complexity protein analysis in crosslinking mass spectrometry analysis by improving signal-to-noise and will assess this in direct comparison to standard crosslinkers.

• DFG Programme: Research Grants