Proteoform-Resolved Interaction Mapping of Target Protein Complexes in Living Cells via Tracelessly Releasable Cross-Linking

Abstract

Proteoform-resolved interaction analysis is critical for understanding the functional diversity of multiple proteoforms from the same gene and their complexes. However, current technologies are still limited in resolving in vivo interactions at the proteoform level. In vivo cross-linking followed by bottom-up mass spectrometry (MS) can provide interaction sites but loses proteoform information, whereas top-down MS can characterize proteoform composition but struggles with cross-linked spectral complexity. Herein, we developed a strategy based on tracelessly releasable cross-linking coupled with MS to decipher proteoform-resolved interactions in target protein complexes in living cells. First, we designed a novel cross-linker, bis(N-hydroxysuccinimide) (2,2’-(ethylenebissulfonyl) diethanol) dicarbonate (DSODSE), enabling not only in vivo cross-linking to fix interactions in living cells but also traceless release of cross-linked proteoforms at pH ≥ 8.0 to address the challenges of proteoform identification caused by cross-linking. Benefited from such a cross-linker, an in-depth accurate identification of proteoform components was achieved via in vivo cross-linking, enrichment of target complexes, traceless release, followed by top-down MS. Meanwhile, the cross-linked peptides were analyzed with the identified proteoforms as the database, and the proteoform-resolved interactions were obtained by matching the cross-links to the specific sequence of proteoforms. By this strategy, we deciphered the in vivo proteoform-resolved interaction network of α-syn complexes. To our best knowledge, our work achieves the first delineation of the in vivo proteoform−proteoform interaction network of target protein complexes, providing a powerful tool for understanding the functional diversity of proteoforms and their complexes in living cells.