An acidic residue reactive and disulfide bond-containing cleavable cross-linker for probing protein 3D structures based on electrochemical mass spectrometry

Cross-linking mass spectrometry (XL-MS) has attracted broad attention because of the capability to probe three-dimensional structure of proteins. Up to now, several amine-reactive cross-linkers have been developed for characterization of proteins and protein complexes. However, spatial information retrieved by XL-MS is still limited, partly because the strategies using an acidic residue reactive cross-linker have been rarely reported. In this paper, an acidic residue (e.g. aspartic acid, glutamic acid)-specific, disulfide bond-containing, cleavable cross-linker with a length of 13.1 Å, named 3,3′-dithiobis(propanoic dihydrazide), was presented for the first time. In addition, a novel approach using the cross-linker was proposed for unambiguous characterization of peptides and proteins with disulfide bonds. After cross-linked, the peptides could be electrochemically reduced, then characterized by high performance liquid chromatography mass spectrometry. For demonstration, the approach has been adopted to characterize the emideltide, insulin, and myoglobin, of which four pairs of intrachain cross-links have been successfully identified in myoglobin. The results showed that the cross-links displayed predictable fragmentation pattern upon collision induced dissociation (CID), thus admitting simplifying data analysis. In summary, this work introduces a novel type of cross-linker utilized for cross-linking and a new strategy to XL-MS technology for comprehensively understanding the three-dimensional structure of proteins.