Quaternary Diamines as Mass Spectrometry Cleavable Crosslinkers for Protein Interactions

Mapping protein interactions and their dynamics is crucial to defining physiologic states, building effective models for understanding cell function, and to allow more effective targeting of new drugs. Crosslinking studies can estimate the proximity of proteins, determine sites of protein-protein interactions, and have the potential to provide a snapshot of dynamic interactions by covalently locking them in place for analysis. Several major challenges are associated with the use of crosslinkers in mass spectrometry, particularly in complex mixtures. We describe the synthesis and characterization of a MS-cleavable crosslinker containing cyclic amines, which address some of these challenges. The DC4 crosslinker contains two intrinsic positive charges, which allow crosslinked peptides to fragment into their component peptides by collision-induced dissociation (CID) or in-source decay. Initial fragmentation events result in cleavage on either side of the positive charges so crosslinked peptides are identified as pairs of ions separated by defined masses. The structures of the component peptides can then be robustly determined by MS3 because their fragmentation products rearrange to generate a mobile proton. The DC4 crosslinking reagent is stable to storage, highly reactive, highly soluble (1 M solutions), quite labile to CID, and MS3 results in productive backbone fragmentation.