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

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.