A novel cross-linking strategy for aromatic residues

Chemical cross-linking in combination with mass spectrometry (CXMS) has been developed into a powerful method for mapping protein structures, dynamics and interaction networks including molecular interfaces in protein-protein and protein-nucleic acid complexes. Although many cross‑linkers have been developed in last two decades, majority of CXMS analyses still utilizes lysine-specific cross-linking reagents based on N-hydroxysuccinimide esters. Other cross-linking reagents have only limited use for various reasons such as low reactivity, low occurrence of targeted residues, unwanted side products etc. In this study we show a new generation of cross-linkers based on recently published Fast FluoroAlkylation of Proteins (FFAP) technology that enables targeting of aromatic amino acids residues.

Cross-linking reaction is based on two step FFAP mechanism which includes activation of hypervalent iodine using metal ions or lewis acid and subsequent attack of aromatic residues by the resulting radical. The studied proteins were analyzed by bottom up approach using mass spectrometry (timsTOF SCP, Bruker Daltonics) where samples were digested by trypsin protease, separated on reverse phase column online coupled to mass spectrometer.

The results on several protein models such as horse heart myoglobin clearly demonstrate the potential of our novel cross-linking strategy. The cross-links of aromatic residues generated by fluoroalkyl radicals nicely correspond with protein crystal structures of studied protein and provide information not attainable by lysine-specific cross-links. Our data lead to assumption the cross-linker based on fluoroalkyl radicals could be use as fast and powerful method to study protein structure and dynamics in solution.

This work was mainly supported by the Czech Science Foundation (grant number 22-27695S) and European Commission H2020 (The European Proteomics Infrastructure Consortium providing access EPIC-XS – project agreement No. 823839).