Facilitating In Situ Cross-Linking and Mass Spectrometry by Antibody-Based Protein Enrichment.

Abstract

Cross-linking of living cells followed by mass spectrometry identification of cross-linked peptides (in situ CLMS) is an emerging technology to study protein structures in their native environment. One of the inherent difficulties of this technology is the high complexity of the samples following cell lysis. Currently, this difficulty largely limits the identification of cross-links to the more abundant proteins in the cell. Here, we describe a targeted approach in which an antibody is used to purify a specific protein-of-interest out of the cell lysate. Mass spectrometry analysis of the protein material that binds to the antibody can then identify considerably more cross-links on the target protein. By using an antibody against the CCT chaperonin, we identified over 200 cross-links that provide in situ evidence for the subunit arrangement of the CCT particle and its interactions with prefoldin. Similar targeting with an antibody against tubulin provided in situ evidence for the structure of the microtubule. Finally, the approach was also successful in identifying cross-links within a protein that expresses at a low level. These results demonstrate the general utility of antibody-based sample simplification for in situ CLMS and greatly expand the scope of protein systems that are amenable to in situ structural studies.