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

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

Proceedings of the National Academy of Sciences of the United States of America (2014-06-19)
Alexander Leitner, Lukasz A Joachimiak, Pia Unverdorben, Thomas Walzthoeni, Judith Frydman, Friedrich Förster, Ruedi Aebersold
摘要

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.

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