Leitner, A. et al. Proc. Natl. Acad. Sci. USA 111, 9455–9460 (2014).

The combination of chemical cross-linking and mass spectrometry is proving useful for helping to elucidate the structure of protein complexes. With mass spectrometry, the locations of cross-linked sites can be unambiguously identified, providing spatial information about the arrangement of subunits in a complex. Current chemical cross-linking reagents typically target the primary amine moieties found on lysine residues. Leitner et al. now show that a reaction combining homobifunctional dihydrazide molecules with a coupling reagent can be used to cross-link the acidic residues aspartic acid and glutamic acid. As test cases, they studied the chaperonin TRiC and the 26S proteasome complexes. Not only does the acidic residue cross-linking reaction provide an alternative to lysine cross-linking, it provides orthogonal distance restraints, which expands the amount of structural information that can be obtained.