Ping-Pong Protons: How Hydrogen-Bonding Networks Facilitate Heterolytic Bond Cleavage in Peptide Radical Cations

Electron capture and electron transfer dissociation (ECD/ETD) tandem mass spectrometry (MS/MS) are commonly employed techniques for biomolecular analysis. The ECD/ETD process predominately cleaves N–Cα peptide backbone bonds, leading to primary sequence information complementary to other mass spectrometry techniques. Despite frequent laboratory use, the mechanistic underpinnings surrounding N–Cα bond cleavage remain debated. While the majority of mechanisms assume a homolytic bond rupture, we recently showed that heterolytic cleavage is also thermodynamically viable. For a cleavage of this type to be feasible, the charge separation created upon breaking of the N–Cα backbone bond must be quickly annihilated. In this work, we show, using density functional computations, that specific hydrogen-bonding motifs and structural rearrangements involving proton transfers stabilize the transition state associated with heterolytic cleavage and eliminate the ensuing charge separation from the final product fragments. The movement of protons can occur either directly from the z- to c-fragment or in a more complex manner including a ping-pong-type mechanism. The nature of these diverse hydrogen-bonding motifs reveals that not only those functional groups proximate to the bond rupture site, but also the entire global chemical environment, play important roles in backbone cleavage characteristic of ECD/ETD MS/MS. For doubly charged systems, both conformation and electron localization site dictate which of the two fragments retains the final positive charge.