Chemical crosslinking combined with mass spectrometry is a useful tool for studying the topological organization of multiprotein interactions, but it is technically challenging to identify peptides involved in a crosslink using tandem mass spectrometry (MS/MS) due to the presence of product ions originating from both peptides within the same crosslink. We have previously developed a novel set of collision-induced dissociative chemical crosslinking reagents (CID-CXL reagents) that incorporate a labile bond within the linker which readily dissociates at a single site under low-energy collision-induced dissociation (CID) to enable independent isolation and sequencing of the crosslinked peptides by traditional MS/MS and database searching. Alternative low-energy CID events were developed within the in-source region by increasing the multipole DC offset voltage (ISCID) or within the ion trap by increasing the collisional excitation (ITCID). Both dissociation events, each having their unique advantages, occur without significant backbone fragmentation to the peptides, thus permitting subsequent CID to be applied to these distinct peptide ions for generation of suitable product ion spectra for database searching. Each approach was developed and applied to a chemical crosslinking study involving the N-terminal DNA-binding domain of AbrB (AbrBN), a transition-state regulator in Bacillus subtilis. A total of thirteen unique crosslinks were identified using the ITCID approach which represented a significant improvement over the eight unique crosslinks identified using the ISCID approach. The ability to segregate intrapeptide and interpeptide crosslinks using ITCID represents the first step towards high-throughput analysis of protein-protein crosslinks using our CID-CXL reagents.
Copyright (c) 2007 John Wiley & Sons, Ltd.