Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping - A novel approach to assess intermolecular protein contacts

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical crosslinkin g with differential MALDI-MS analyses. ParR dimers were modified in vitro w ith the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionat e) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cros s-linked ParR dimers in the presence and absence of a thiol reagent strongl y supported a "head-to-tail" arrangement of the monomers in the dimeric com plex. Glycoprotein fusion constructs CD28-IgG and CD80-F-ab were cross-link ed in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in si tu with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent ). The data revealed the presence of an intermolecular cross-link between t he receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-F-ab, The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+/- thi ol reagent) enabled localization of the interface region(s) of the complexe s studied and clearly demonstrates the utility of such an approach to obtai n structural information on interacting noncovalent complexes.