Direct Observation of Metastable Fragment Ions in Ultraviolet Photodissociation of Ubiquitin

Abstract

Ultraviolet photodissociation (UVPD) of proteins is known to exhibit conformation-dependent fragmentation patterns, but direct structural evidence linking precursor protein and fragment ions has been limited. Here, we apply tandem trapped-ion mobility spectrometry/tandem-mass spectrometry to compare collision cross sections of UVPD fragment ions generated from distinct conformers of ubiquitin. Under the high-pressure (∼4 mbar) and low-photon density (∼10 μJ laser pulse energies) conditions employed here, UVPD produces predominantly [b + 2] and [y - 2] ions at proline residues, consistent with direct bond cleavage from the electronically excited state. Our data show that these ions can retain a clear structural relationship to the precursor conformation: UVPD of compact, native-like ubiquitin yields fragments with collision cross sections ∼20% smaller than the corresponding ions produced from extended precursors or by collision-induced dissociation. Further, these compact UVPD fragments are kinetically trapped in metastable conformations, with substantial barriers preventing relaxation toward energetically favored gas-phase structures. We attribute this behavior to limited vibrational energy deposition per absorbed 213 nm photon combined with rapid collisional cooling, which suppress cumulative thermal activation and disfavor statistical fragmentation pathways, leaving direct excited-state dissociation as the dominant observable process. Together with prior UVPD studies on holo-myoglobin, our results suggest that UVPD fragments can retain aspects of their precursor tertiary structure.