Capturing Polyradical Protein Cations after an Electron Capture Event: Evidence for their Stable Distonic Structures in the Gas Phase

We report on the formation and “capture” of polyradical protein cations after an electron capture event. Performed in a unique electron-capture dissociation (ECD) instrument, these experiments can generate reduced forms of multiply protonated proteins by sequential charge reduction using electrons with ~1 eV. The true structures of these possible polyradicals is considered: Do the introduced unpaired electrons recombine quickly to form a new two-electron bond, or do these unpaired electrons exist as radical sites with appropriate chemical reactivity? Using an established chemical probe—radical quenching with molecular oxygen—we demonstrate that these charge-reduced protein cations are indeed polyradicals that form adducts with up to three molecules of oxygen (i.e., tri-radical protein cations) that are stable for at least 100 ms.