DSSBU: novel cross-linker tested on bovine serum albumin and human hemoglobin

Protein cross-linking has assumed an irreplaceable role in structural proteomics. Recently, significant efforts have been made to develop novel MS-cleavable reagents. These cross-linkers enhance the reliability of cross-link identification. Presently, only water-insoluble MS-cleavable cross-linkers are commercially available. However, the comprehensive analysis of the various chemical and structural motifs inherent in proteins depends on the targeting of different protein sites with varying degrees of hydrophilicity. We introduce a new MS-cleavable cross-linker called disulfodisuccinimidyl dibutyric urea (DSSBU), which we have developed in-house. DSSBU contains an N-hydroxysulfosuccinimide (sulfo-NHS) reactive group. It therefore serves as a water-soluble counterpart to the commercially available and widely used disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers—bis[sulfosuccinimidyl] suberate (BS3), disuccinimidyl suberate (DSS), DSBU and DSSBU on bovine serum albumin. We also compared efficacy of DSBU and DSSBU on human hemoglobin. Our results demonstrate that the superior water solubility of DSSBU enables to avoid the usage of polar solvents such as DMSO but still maintaining the effectivity of MS-cleavable crosslinker such as DSBU.