Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

Alexander Leitner, Lukasz A. Joachimiak, Pia Unverdorben, Thomas Walzthoeni, Judith Frydman, Friedrich Förster, Ruedi Aebersold

Research output: Contribution to journalArticlepeer-review

158 Scopus citations


The study of proteins and protein complexes using chemical crosslinking 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.

Original languageEnglish (US)
Pages (from-to)9455-9460
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number26
StatePublished - Jul 1 2014
Externally publishedYes

ASJC Scopus subject areas

  • General


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