biostudies-literatureRenbarger EANIGMS NIH HHS117570https://www.ebi.ac.uk/biostudies/studies/S-EPMC12798706biostudies-literatureUnknown521Cleavable 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 International journal of mass spectrometryEvaluation of Cleavable Crosslinking for Characterization of Proteoform Structural Differences by Top-Down Mass Spectrometry.PMC12798706R35 GM151251Renbarger EAWebb IKfalseEvaluation 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 2026-01-01T00:00:00Z2026 Mar2026-06-06T17:30:14.57Z2026-06-03T03:10:39.253ZS-EPMC127987064153693210.1016/j.ijms.2026.117570