DSBU (disuccinimidyl dibutyric urea), or BuUrBU, is a mass-spectrometry (MS) -cleavable and membrane-permeable cross-linker that contains an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of an 11-atom spacer arm. The cross-linker facilitates analysis of protein structure and complex interactions using mass spectrometry. DSBU has similar reactivity to DSS, but contains a urea linker that can be cleaved in the gas phase during tandem MS (MS/MS) using collision-induced dissociation (CID). The ability to cleave cross-linked peptides during MS/MS enables MS3 acquisition methods, which facilitate peptide sequencing using traditional database search engines. The MS cleavage of DSBU also generates diagnostic ion doublets during MS2, which enables identification of cross-linked peptides from dead-end modifications and searching using novel database search engines such as MeroX or XlinkX.
Features of DSBU: • Reactive group: NHS ester (both ends) • Amine-reactive NHS ester reacts rapidly with any primary amine-containing molecule • Membrane permeable, allowing for intracellular crosslinking • High-purity, crystalline reagent can be used to create high-purity conjugates • MS-cleavable • Water-insoluble (dissolve first in DMF or DMSO) • Purity: > 90% by quantitative NMR (the highest standard in crosslinking purity)
Chemical crosslinking in combination with mass spectrometry is a powerful method to determine protein-protein interactions. This method has been applied to recombinant and native protein complexes, and more recently, to whole cell lysates or intact unicellular organisms in efforts to identify protein-protein interactions on a global scale. Both traditional non-cleavable and MS-cleavable cross-linkers provide insight into the identification of protein-protein interaction sites, but MS-cleavable cross-linkers are advantageous due to their ability to be cleaved using different types of gas-phase fragmentation methods (e.g., CID, HCD, ETD, and EThcD) and levels of tandem mass spectrometry (e.g., MS2 and MS3) in order to improve identification of cross-linked peptides.
References 1. Müller MQ, Dreiocker F, Ihling CH, Schäfer M, Sinz A. Cleavable cross-linker for protein structure analysis: reliable identification of cross-linking products by tandem MS. Anal Chem. 2010 Aug 15;82(16):6958-68. 2. Arlt C, Götze M, Ihling CH, Hage C, Schäfer M, Sinz A. Integrated Workflow for Structural Proteomics Studies Based on Cross-Linking/Mass Spectrometry with an MS/MS Cleavable Cross-Linker. Anal Chem. 2016 Aug 16;88(16):7930-7.