Photocleavable crosslinker design, synthesis and analysis for enhanced crosslinking mass spectrometry methods
Final Report Abstract
This research project developed new chemical tools and workflows to study the structures of proteins and their interactions. The approach is based on a technique known as crosslinking mass spectrometry (crosslinking MS), which allows researchers to “freeze” interactions between proteins by chemically linking them together and then identifying the linked regions through mass spectrometry. A central challenge in this field is the identification of crosslinked peptide pairs, especially when using chemical linkers that fragment under the same conditions as the peptides themselves. To overcome this, the project introduced a new class of UV-cleavable crosslinkers (UCCLs) that can be selectively fragmented using ultraviolet light, independent of the peptide fragmentation that is necessary for identifying peptides. This leads to much cleaner data and more confident identification of the crosslinked peptides. The project also developed new computational algorithms to make use of this light-based cleavage in automated data acquisition workflows. The result is a streamlined and accessible approach that was successfully tested in complex protein mixtures derived from E. coli cells. Importantly, all experiments were conducted on commercially available mass spectrometers, meaning that other labs can adopt these methods without the need for hardware modifications. The new crosslinkers and methods were published in peer-reviewed journals and are already influencing instrument and software development at commercial suppliers. This shows not only academic impact but also potential for future applications in biotechnology and structural biology. Understanding how proteins are structured and how they interact is essential for uncovering how cells function, how diseases develop, and how new therapies can be designed. Crosslinking MS is one of the few techniques that can provide structural information directly from complex biological samples. This work contributes to the broader effort to map protein structures and interactions at scale, supporting both fundamental research and biomedical discovery. Future work is now focused on expanding this strategy using photoactivatable, heterobifunctional crosslinkers, which offer even greater utility for large-scale studies.
Publications
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Cleavable Cross-Linkers Redefined by a Novel MS3-Trigger Algorithm. Analytical Chemistry, 95(42), 15461-15464.
Kolbowski, Lars; Fischer, Lutz & Rappsilber, Juri
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Light-Induced Orthogonal Fragmentation of Crosslinked Peptides. JACS Au, 3(8), 2123-2130.
Kolbowski, Lars; Belsom, Adam; Pérez-López, Ana M.; Ly, Tony & Rappsilber, Juri
