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

Abstract

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 crosslinking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,39-dithio-bis~succinimidylproprionate!~DTSSP!, proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-F ab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping~6 thiol reagent!. The data revealed the presence of an intermolecular cross-link between the 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~6 thiol reagent! enabled localization of the interface region~s! of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.

Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

“…One major shortcoming of chemical cross-linking approaches, however, is the tremendous sample complexity attributable to the wide variety of cross-linked products that can be created during the cross-linking reaction. To circumvent this disadvantage, a number of different strategies have been developed that have in common to facilitate identification of cross-linker containing species, e.g., by employing isotope-labeled cross-linkers [6,7], isotope-labeled proteins [8], cleavable cross-linkers [9], fluorogenic crosslinkers [10], or cross-linkers creating a characteristic marker ion during MS/MS analysis [11]. Another strategy to selectively enrich cross-linker containing species by using trifunctional cross-linkers containing a biotin moiety has been described recently [12][13][14].…”

Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-linking, and Mass Spectrometry

“…This problem has been partially addressed by "tagging" methodologies that allow rapid visual MS location of cross-linked species within complex peptide mixtures (34,(37)(38)(39). For example, the use of a 1:1 mixture of undeuterated and deuterated (d 0 /d 4 -labeled) cross-linking reagent readily allows mass spectrometric detection of all cross-linked species by the presence of d 0 /d 4 -isotope tags (39).…”

“…This results in cross-linked peptide species being tagged that do not yield useful information on intermolecular interactions, such as intramolecular crosslinked peptides or peptides modified by partially hydrolyzed cross-linking reagent (30,40). Moreover, these tagging methodologies make cross-linking studies of oligomeric proteins, such as IL-6 D , ambiguous because identical cross-linked peptides can in principle arise from inter-or intramolecular origins (38).…”

Trapping Transient Protein–Protein Interactions in Polyketide Biosynthesis

“…Chemical cross-linking of proteins with variable binding affinities provides a means of assaying critical contact sites (1)(2)(3)(4)(5)(6). For certain specialized cases, natural enzymatic activities may be exploited to serve as highly selective covalent stabilization.…”