Advances in crosslinking chemistry and proximity-enabled strategies: deciphering protein complexes and interactions

Mass spectrometry, coupled with innovative crosslinking techniques to decode protein conformations and interactions through uninterrupted signal connections, has undergone remarkable progress in recent years. It is crucial to develop selective crosslinking reagents that minimally disrupt protein structure and dynamics, providing insights into protein network regulation and biological functions. Compared to traditional crosslinkers, new bifunctional chemical crosslinkers exhibit high selectivity and specificity in connecting proximal amino acid residues, resulting in stable molecular crosslinked products. The conjugation with specific amino acid residues like lysine, cysteine, arginine and tyrosine expands the XL-MS toolbox, enabling more precise modeling of target substrates and leading to improved data quality and reliability. Another emerging crosslinking method utilizes unnatural amino acids (UAAs) derived from proximity-enabled reactivity with specific amino acids or sulfur-fluoride exchange (SuFEx) reactions with nucleophilic residues. These UAAs are genetically encoded into proteins for the formation of specific covalent bonds. This technique combines the benefits of genetic encoding for live cell compatibility with chemical crosslinking, providing a valuable method for capturing transient and weak protein-protein interactions (PPIs) for mapping PPI coordinates and improving the pharmacological properties of proteins. With continued advancements in technology and applications, crosslinking mass spectrometry is poised to play an increasingly significant role in guiding our understanding of protein dynamics and function in the future.