Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.
- Leitner A Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland; leitner@imsb.biol.ethz.ch aebersold@imsb.biol.ethz.ch.
- Joachimiak LA Department of Biology, Stanford University, Stanford, CA 94305;
- Unverdorben P Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany; and.
- Walzthoeni T Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
- Frydman J Department of Biology, Stanford University, Stanford, CA 94305;
- Förster F Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany; and.
- Aebersold R Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;Faculty of Science, University of Zurich, 8057 Zurich, Switzerland leitner@imsb.biol.ethz.ch aebersold@imsb.biol.ethz.ch.
- 2014-06-19
Published in:
- Proceedings of the National Academy of Sciences of the United States of America. - 2014
Acids
Chaperonins
Cross-Linking Reagents
Lysine
Mass Spectrometry
Molecular Structure
Multiprotein Complexes
Proteins
Proteomics
English
The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.
- Language
-
- English
- Open access status
- bronze
- Identifiers
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- DOI 10.1073/pnas.1320298111
- PMID 24938783
- Persistent URL
- https://folia.unifr.ch/global/documents/40457
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