Near-Edge Soft X-ray Absorption Mass Spectrometry of Protonated Melittin

Abstract

We have investigated the photoionization and photofragmentation yields of gas-phase multiply protonated melittin cations for photon energies at the K-shell absorption edges of carbon, nitrogen, and oxygen. Two similar experimental approaches were employed. In both experiments, mass selected [melittin+qH] (q=2-4) ions were accumulated in radiofrequency ion traps. The trap content was exposed to intense beams of monochromatic soft X-ray photons from synchrotron beamlines and photoproducts were analyzed by means of time-of-flight mass spectrometry. Mass spectra were recorded for fixed photon energies, and partial ion yield spectra were recorded as a function of photon energy. The combination of mass spectrometry and soft X-ray spectroscopy allows for a direct correlation of protein electronic structure with various photoionization channels. Non-dissociative single and double ionization are used as a reference. The contribution of both channels to various backbone scission channels is quantified and related to activation energies and protonation sites. Soft X-ray absorption mass spectrometry combines fast energy deposition with single and double ionization and could complement established activation techniques. Graphical Abstract ᅟ.

Breaking inversion symmetry by protonation: experimental and theoretical NEXAFS study of the diazynium ion, N2H+

“…It is also clear though that the superposition is delicately dependent on the electronic energy separation of the two p* excited states. All these effects are subtle and can probably only be resolved in simple protonated species as N 2 H + studied here, whereas in XAS of protonated peptides or proteins [55][56][57][58] these features would probably not be observed.…”

Development of Ultrafast X-ray Free Electron Laser Tools in (Bio)Chemical Research

“…Prolongating from the already presented techniques of combining X-ray scattering techniques like diffuse X-ray scattering with pressure jump, temperature jump, electric field modulations, and structural freezing methods or, on the chemical modulation side, with, rapid mixing or photo-switching methods and their successful application to biophysical questions [90, 94-103, 115, 116, 118, 121] or the application of core hole clock and multidimensional X-ray spectroscopy techniques towards aqueous and pre-biotics questions [91][92][93][104][105][106][107][108] or novel liquid jet developments for biophysical research [120], in X-ray spectroscopy various other coupling techniques have been successfully proven. The combination of synchrotrons or free-electron laser radiation with techniques, such as electrospray ionization mass spectrometry, allows deriving entirely novel experimental techniques for investigating macromolecules [109][110][111][112][113][114][115][116][117][118][119][120][121]. E.g.…”

“…17.16). Electrospray ionization introduces biomolecular ions from solution into the gas phase, allowing for studies of molecular systems in a well-defined state [109,[111][112][113][114]119].…”

“…The very fast loss of side chains thus efficiently cools the residual peptide, and may enable the survival of early functional peptide substructures under photon irradiation. This dissociation is mainly caused by photoabsorption in the peptide backbone, which seems to trigger ultrafast charge migration to the side chains, without leading to fragmentation to the backbone itself [111][112][113][114]119].…”

“…Additional structural and dissociation dynamics within the molecules can be extracted from the NEXAMS information of the different individual ionization and dissociation products. Moreover, the gas-phase NEXAMS method can be sensitive to the secondary structure of proteins as shown in the study of melittin [113].…”