title: Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.
abstract: 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.
authors.affiliation: 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.
authors.forename: Alexander
authors.initials: A
authors.lastname: Leitner
authors.affiliation: Department of Biology, Stanford University, Stanford, CA 94305;
authors.forename: Lukasz A
authors.initials: LA
authors.lastname: Joachimiak
authors.affiliation: Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany; and.
authors.forename: Pia
authors.initials: P
authors.lastname: Unverdorben
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
authors.forename: Thomas
authors.initials: T
authors.lastname: Walzthoeni
authors.affiliation: Department of Biology, Stanford University, Stanford, CA 94305;
authors.forename: Judith
authors.initials: J
authors.lastname: Frydman
authors.affiliation: Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany; and.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Förster
authors.affiliation: 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.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000143:Acids / Q000032:analysis*; D018833:Chaperonins / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D046912:Multiprotein Complexes / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
chemical_list: D000143:Acids; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D011506:Proteins; D018833:Chaperonins; D008239:Lysine