Robust Chemical Cross-linking of Proteins and Protein Complexes via SuFEx Click Chemistry

Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) has emerged as a highly efficient tool for elucidating the structures of proteins and protein complexes. This technology offers valuable insights into protein conformations, interactions, and dynamics, which are crucial for understanding biological functions and diseases. A pressing need in CXMS is to develop new chemical cross-linkers with expanded reactivity and unique physicochemical properties, which will broaden the cross-linking coverage and lead to more precise modeling of protein structures with improved data quality and reliability. Herein, we developed sulfonyl fluoride (SF)-containing homodimeric cross-linkers based on sulfur-fluoride exchange (SuFEx) click chemistry. These cross-linkers group at a distinct location in chemical space. Reactions on model peptides, proteins, and cell lysates demonstrated their robust reactivity toward tyrosine and lysine residues. Notably, SF cross-linking aligns well with known protein structures and enables the generation of 3D models for proteins and protein complexes with no prior experimental data. These results highlight the potential of SuFEx click chemistry to expand the CXMS toolbox and provide complementary information that cannot be accessed by current chemical cross-linking reagents.