Sheng Zhang

　　Director of Proteomics and Mass Spectrometry Facility; Sr. Research Associate for Institute of Biotechnology, Cornell University, USA

　　The majority of proteins execute their functions in the form of protein complexes, which are important parts of well-regulated pathways and networks playing critical roles in regulating integral biological processes. Investigations of protein complexes and specific protein-protein interactions is a key step targeted to understanding protein-protein interaction networks. But, it represents a highly challenging area from both sample preparation strategy and analytical perspectives. Cross linking mass spectrometry (XL-MS) is increasingly becoming a powerful analytical tool providing a vital insight into the structure and interactions of proteins/protein complexes. By freezing transient interactions through the formation of covalent bonds, it can capture temporal protein interactions yielding information on the identities and spatial orientations of interacting proteins simultaneously.

　　Recently several types of MS-cleavable cross-linkers have been developed to facilitate MS identification of crosslinked peptides. Disuccinimidyl sulfoxide (DSSO) is one of the cleavable cross-linkers containing symmetric MS-labile C-S bonds as preferential cleavage sites upon MS/MS fragmentation of cross-linked precursors and producing two pairs of signature fragment ions from each xlinks. This unique feature of signature fragment ions allows for “targeted mass difference” selection in Orbitrap Fusion analysis for unambiguous identification of cross-linked peptides by MS3 analysis and conventional database searching tools (XlinkX and Proteome Discoverer 2.2).   

　　This DSSO-based XL-MS using MS2/M3 approach was initially tested for BSA and bovine GDH. About 62 and 19 unique xlinks in BSA and GDH respectively were identified. The identified xlinks were confirmed by manual inspection of 3D structures containing both intra- and inter- subunit xlinks. This XL-MS approach was used for analysis of transmembrane complexes that mediate bacterial chemotaxis. The signaling network of bacterial chemotaxis comprises complex interactions within a large multi-component transmembrane assembly (CheA, CheW and Receptor), whose detailed architecture is unknown. CheA is a histidine kinase, a key sensor of chemotaxis pathway containing 5 domains (P1-P5). All 5 individual domains’ structures were solved, but no domain topology is available. The domain mapping results (8 xlinks) identified by XL-MS provide first evidence that a proximate interaction of the P4 domain with ATP binding pocket to the P1 domain containing His45 being phosphorylated from ATP hydrolysis. Furthermore, the XL-MS was applied for facilitating 3D structural modeling of the recommended “rCheA, rCheW and rReceptor” ternary complex. We have identified 31 xlinks within 3 individual proteins and 11 xlinks between the two different proteins. The results support the electron spin resonance models for the solution structure of the ternary complex.