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

Published online by Cambridge University Press:  01 August 2000

KEIRYN L. BENNETT ,
MARTIN KUSSMANN ,
PER BJÖRK ,
MAGDALENA GODZWON ,
MARIE MIKKELSEN ,
POUL SØRENSEN  and
PETER ROEPSTORFF
KEIRYN L. BENNETT
Affiliation:
Department of Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
MARTIN KUSSMANN
Affiliation:
Department of Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark Current address: Cerbios-Pharma S.A., Via Pian Scairolo 6, CH-6917 Barbengo, Switzerland.
PER BJÖRK
Affiliation:
Active Biotech Research AB, Scheelevägen 22, SE-220 07 Lund, Sweden
MAGDALENA GODZWON
Affiliation:
Active Biotech Research AB, Scheelevägen 22, SE-220 07 Lund, Sweden
MARIE MIKKELSEN
Affiliation:
Department of Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
POUL SØRENSEN
Affiliation:
Active Biotech Research AB, Scheelevägen 22, SE-220 07 Lund, Sweden
PETER ROEPSTORFF
Affiliation:
Department of Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
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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 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.

Keywords

cross-linkingDNA-binding proteinglycoproteinmass spectrometrynoncovalent interactions
Type
Research Article
Information
Protein Science , Volume 9 , Issue 8 , August 2000 , pp. 1503 - 1518
Copyright
© 2000 The Protein Society

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