Thianthrenium Chemistry for Identification of Protein–Protein Interactions in Cells

We developed a synthetically readily accessible dicationic thianthrenium reagent for reliable identification of protein-protein interactions (PPIs) <i>in cellulo</i>. Cysteine (Cys)-selective Michael addition to <i>in situ</i> generated alkenyl thianthrenium salts leads to formation of reactive episulfonium intermediates on protein surfaces that covalently trap all nucleophilic amino acids of interacting proteins. Short and chemically stable ethylene linkages are introduced in a single step and thereby provide short distance restraints for reliable modeling of PPI interfaces. The fundamental advance described in this work is the development of a reagent for identification of low-abundant interprotein cross-links via episulfonium chemistry enabled by the introduction of an alkyne tag for downstream enrichment of cross-linked products without an extension of the linkage length between the two cross-linked amino acids. Based on the fast single-step cross-linking, we show that our method allows for more accurate structure prediction of reported PPIs compared to data produced by commonly used bifunctional cross-linking reagents. We further demonstrate that our approach enables identification of previously undocumented PPIs in human cancer cells by targeting native Cys residues and that the obtained short distance restraints provide valuable information for modeling of PPI surfaces.