Proton-Transfer Reactions Modulate Protein Ion Structure to Yield Altered Backbone Cleavage Pathways in Top-Down Mass Spectrometry

Abstract Proton-transfer reaction (PTR) has emerged as a powerful tool for top-down proteomics. It can mitigate spectral congestion and improve the signal-to-noise ratio of electrospray ionization (ESI)-generated intact proteins. We hypothesized that charge removal during PTR of Coulombically unfolded, denatured ESI ions induces rapid gas-phase isomerization and refolding, producing charge-state-dependent conformers with altered fragmentation behavior. Here, we test this hypothesis by systematically comparing fragmentation patterns of three model proteins, apomyoglobin, apocalmodulin, and histone H1, with and without PTR using high-resolution top-down mass spectrometry. Highly charged precursor ions were isolated, subjected to PTR, fragmented, and benchmarked against ions of the same charge state generated directly from ESI; ions generated under native-like conditions were also examined. Quantitative analysis of assigned fragment ions and log2 fold-change heatmaps reveal distinct fragmentation patterns, with some regions showing protection from fragmentation, while others yield newly observed or substantially enhanced fragment ions. Notably, characteristic fragmentation signatures around proline and aspartic acid residues further demonstrate structural differences. These results show that protein relaxation after PTR generates different gas-phase ion structures than are formed during ESI and explain, alongside mobile proton theory, why charge reduction produces different sets of fragment ions in top-down MS/MS, but can also yield fragmentation sites otherwise unavailable for higher-charged counterparts and the same charge states generated without PTR.