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

• Published in: Protein science 2000-08, Vol.9 (8), p.1503-1518
• Main Authors: BENNETT, KEIRYN L., KUSSMANN, MARTIN, BJÖRK, PER, GODZWON, MAGDALENA, MIKKELSEN, MARIE, SØRENSEN, POUL, ROEPSTORFF, PETER
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; B7-1 Antigen - chemistry; B7-1 Antigen - metabolism; Bacterial Proteins; Binding Sites; CD28 Antigens - chemistry; CD28 Antigens - metabolism; Cross-Linking Reagents; cross‐linking; Cysteine - chemistry; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; DNA‐binding protein; Electrophoresis, Polyacrylamide Gel; glycoprotein; Lysine - chemistry; mass spectrometry; Molecular Sequence Data; noncovalent interactions; Peptide Mapping - methods; Repressor Proteins - chemistry; Repressor Proteins - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.9.8.1503

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 cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3′-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-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (± 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-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (± 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.