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 approach is the high complexity of the samples following cell lysis. 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 pulls a specific protein-of-interest out of the lysate. Mass spectrometry analysis of the protein material that binds to the antibody can then identify considerably more cross-links on the antibody target and its interactors. By using an antibody against the CCT chaperonin, we obtained over two hundred cross-links that provide in situ evidence for the subunit arrangement of CCT and its main interactions with prefoldin. Antibodies against tubulin likewise provided in situ evidence for the structure of the microtubule including the seam. Finally, the approach was also successful in identifying cross-links on a protein expressing at very low amounts (tau in non-neuronal cells). These results demonstrate the general applicability of antibody-based sample simplification for in situ CLMS.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Joanna Zamel  

LAB HEAD: Nir Kalisman

PROVIDER: PXD025099 | Pride | 2022-06-09

REPOSITORIES: Pride