RDD-HCD Provides Variable Fragmentation Routes Dictated by Radical Stability

[Image: see text] Radical-directed dissociation (RDD) is a fragmentation technique in which a radical created by selective 213/266 nm photodissociation of a carbon–iodine bond is reisolated and collisionally activated. In previous RDD experiments, collisional activation was effected by ion-trap collision-induced dissociation (CID). Higher-energy collisional dissociation (HCD) differs from CID both in terms of how ions are excited and in the number, type, or abundance of fragments that are observed. In this paper, we explore the use of HCD for activation in RDD experiments. While RDD-CID favors fragments produced from radical-directed pathways such as a/z-ions and side chain losses regardless of the activation energy employed, RDD-HCD spectra vary considerably as a function of activation energy, with lower energies favoring RDD while higher energies favor products resulting from cleavage directed by mobile protons (b/y-ions). RDD-HCD therefore affords more tunable fragmentation based on the HCD energy provided. Importantly, the abundance of radical products decreases as a function of increasing HCD energy, confirming that RDD generally proceeds via lower-energy barriers relative to mobile-proton-driven dissociation. The dominance of b/y-ions at higher energies for RDD-HCD can therefore be explained by the higher survivability of fragments not containing the radical after the initial or subsequent dissociation events. Furthermore, these results confirm previous suspicions that HCD spectra differ from CID spectra due to multiple dissociation events.