How Native Are Peptides After Activation by Collisions or Photons? A Gas-Phase FRET Study

Native mass spectrometry ionizes biomolecules from aqueous buffered solutions using electrospray ionization. Collisions and lasers are often used to study the structures of such native biomolecular ions. While structural changes upon collisions have been studied in more detail, interactions with photons mostly comprise fragmentation. It remains unclear to what degree biomolecular ions undergo unfolding until cleavage. Here, gas-phase Förster resonance energy transfer (FRET) is used to study fluorescence lifetimes of a 32-residue α-helical peptide to monitor peptide unfolding. Increases in lifetime of up to 1.2 ns per charge are observed for different charge states, showing that a low charge is necessary for peptides to retain a compact structure. Increases in lifetime by up to 0.5 ns are observed upon collisional and laser-based activation and show that the peptide is partially unfolding upon activation. The results contribute to understanding the unfolding dynamics of biomolecules upon activation in mass spectrometry experiments.