Characterization of proteoforms within α-synuclein complexes in living cells by cleavable cross-linking combined with top-down mass spectrometry.

α-Synuclein (α-syn) exists as diverse proteoforms that interact with distinct partners to perform various biological functions. Therefore, top-down mass spectrometry (MS) analysis of proteoform components within α-syn complexes is crucial for understanding their functions. However, the purification of α-syn complexes directly from cell or tissue lysates leads to the loss of weak or transient interacting partners present in the native cellular environment. In vivo cross-linking could fix native interactions before cell lysis, but the top-down MS identification of covalently cross-linked protein complexes is challenging. Herein, to overcome these limitations, a strategy was developed by combining cleavable cross-linking with top-down MS, enabling comprehensive characterization of proteoform composition of α-syn complexes in living cells. Specifically, using dithiobis (succinimidyl-ethyoxyl-propionate) (DSEP), a chemically cleavable cross-linker, the native interactions including transient or weak ones within living cells were fixed via in vivo cross-linking, preventing the loss of interacting partners during subsequent sample preparation. After affinity enrichment of the cross-linked α-syn complexes, the cross-linked proteoforms were decrosslinked via the cleavage of DSEP to facilitate top-down MS analysis. Based on our strategy, 136 proteoforms within α-syn complexes were identified in three biological replicates, which yielded a 2.26-fold increase in proteoform components of α-syn complexes compared with the conventional affinity purification method. Furthermore, the interaction network of α-syn complexes was restructured, yielding detailed proteoform information for each interacting protein, beneficial to uncover more detailed molecular mechanism in the biological processes.