Evaluation of cleavable crosslinking for characterization of proteoform structural differences by top-down mass spectrometry

Cleavable crosslinking has traditionally been employed in bottom-up mass spectrometry to elucidate protein structure and protein-protein interactions through identification of peptides bearing characteristic mass adducts. Here, we demonstrate the application of cleavable crosslinking in top-down mass spectrometry to enhance fragmentation efficiency and enable precise localization of crosslink sites. We first validated this approach using cytochrome c, a well-characterized model protein. Subsequently, we extended top-down cleavable crosslinking to transthyretin, a natively homotetrameric protein exhibiting extensive proteoform heterogeneity, to investigate whether proteoform variations induce structural changes detectable by this method. Our results confirm that cleavable crosslinks can be detected and characterized by top-down mass spectrometry, with crosslinker cleavage under collisional activation significantly enhancing fragmentation. Application to transthyretin (intramolecular crosslinks) yielded complex crosslinking patterns that precluded complete identification of crosslinks. However, the crosslinking data provided valuable information on solvent-accessible residues, functioning effectively as a covalent labeling strategy. This work establishes cleavable crosslinking as a viable chemical crosslinking approach for top-down mass spectrometry applications. • Cleavable crosslinking reveals unique crosslinks in top-down mass spectrometry. • Cytochrome c validation shows added crosslinks and better sequence coverage. • Transthyretin proteoforms show distinct crosslink patterns and structures. • Crosslinking detects subtle differences missed by collision cross-section tests. • Cleaved crosslinkers act as covalent labels for solvent-accessible residues.