New photocrosslinkers for structural biology

We are thrilled to announce the publication of our latest preprint, titled "Design and Characterisation of Photoactivatable and Lysine Reactive o-Nitrobenzyl Alcohol-Based Crosslinkers", now available on bioRxiv. In this work we study the o-nitrobenzyl alcohol photoreactive group and validate its use for structural proteomics.

Photoreactive groups are invaluable tools in structural proteomics, providing reagent-free activation and tem

poral control of protein labelling. Traditional UV-activatable functional groups often produce unstable intermediates and diverse products, posing challenges for large-scale deployment. Our study systematically analyzes the reactivity of ortho-nitrobenzyl alcohol (oNBA) for integration into novel reagents for chemical crosslinking-mass spectrometry.

1. oNBA photochemistry offers a promising alternative to traditional photoactivatable crosslinkers due to its unique specificity towards lysine residues. This specificity enhances could improve crosslink database searching.

2. We synthesized two molecules with oNBA functional groups comprising different substituents, assessing their labelling efficiency against a model protein. Our high power 365 nm irradiation device significantly improved the efficiency of oNBA photolysis, ensuring high labelling yields with short irradiation times.

3. Our studies identified an amide-substituted probe that labels proteins with high efficiency. This optimised oNBA moiety was incorporated into both homo-bifunctional and hetero-bifunctional crosslinkers, resulting in high yields of crosslinked products.

Our findings highlight that optimised oNBA-based reactive groups are viable UV-activated warheads, capable of delivering high labelling yields and efficient protein crosslinking. This unlocks a wealth of potential applications in structural proteomics.

We invite you to read the full preprint on bioRxiv and explore the exciting possibilities these findings present for structural proteomics.