Structure and Charge‐State Dependence of the Gas‐Phase Ionization Energy of Proteins

Multiple Ionization of Free Ubiquitin Molecular Ions in Extreme Ultraviolet Free‐Electron Laser Pulses

“…The pulse energies were attenuated to 0.1 J for single photon absorption conditions. As seen before for soft X-ray [11] and VUV single photon absorption [15] of gas phase protonated proteins, non-dissociative single ionization into [ubi+10H] 11+ (labeled: 11+) is the dominant process, in part accompanied by CO2 loss (m=44, labelled -44). Other large fragment channels are weak.…”

“…From these measurements it was determined that ionization along the peptide backbone followed by fast charge migration towards an aromatic sidechain is the main underlying process. VUV photoionization of larger multiply protonated proteins such as cytochrome c, leads almost exclusively to non-dissociative ionization [15] .…”

“…Ubiquitin has no disulfide bridges and its tertiary structure thus is not very rigid. Tenfold protonation is known to induce substantial structural relaxation driven by Coulomb repulsion of the protonation sites [15] . The gas-phase conformation of [ubi+10H] 10+ can be classified as elongated (with maximum elongation reached for 13-fold protonation) [17] .…”

“…This photon energy well exceeds the binding energy of the deepest O 2s valence orbitals (34.3 eV in glycyl-glycine [20] ). The [ubi+10H] 10+ ionization energy can be extrapolated from the value for [ubi+9H] 9+ (~13.9 eV [15] ) to be 14.2 eV. Well above the ionization threshold, photoionization cross sections are usually reproduced within a few percent by summation of the atomic cross sections [21] .…”

“…This assumption is based on i) the moderate FEL intensity, that leads to only relatively few ionization events per ubiquitin cation and ii) the small increase in protein ionization potential with charge state, that typically amounts to less than 1 eV per charge state, even when structural relaxation is hampered [15] . Assuming Poisson distributed ionization states, the distribution of the degrees of photoionization in the total focal volume, is obtained by mere integration.…”