Soft X‐ray Spectroscopy as a Probe for Gas‐Phase Protein Structure: Electron Impact Ionization from Within

Abstract

Preservation of protein conformation upon transfer into the gas phase is key for structure determination of free single molecules, for example using X‐ray free‐electron lasers. In the gas phase, the helicity of melittin decreases strongly as the protein's protonation state increases. We demonstrate the sensitivity of soft X‐ray spectroscopy to the gas‐phase structure of melittin cations ([melittin+qH]q+, q=2–4) in a cryogenic linear radiofrequency ion trap. With increasing helicity, we observe a decrease of the dominating carbon 1 s–π* transition in the amide C=O bonds for non‐dissociative single ionization and an increase for non‐dissociative double ionization. As the underlying mechanism we identify inelastic electron scattering. Using an independent atom model, we show that the more compact nature of the helical protein conformation substantially increases the probability for off‐site intramolecular ionization by inelastic Auger electron scattering.

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…32,173 Circular dichroism was recently combined with MS for oligonucleotides. 174 With ongoing development of advanced light sources coupled to native MS, 44,175 we expect the possibility of performing spectroscopy analysis of native proteins and complexes for deep structural characterisation to emerge in the near future. The ability to mass-isolate species by MS-based methods could potentially transform structural biology research by offering complementary methods to study non-homogenous samples (e.g., endogenous proteins isolated directly from biological matrices).…”

Inner-shell X-ray absorption spectra of the cationic series NHy+ (y = 0–3)

“…To understand the function of large biomolecules, like amino acids and peptides, one has to study the properties of their fundamental constituents, i.e. their electronic and geometric structure, [1][2][3][4] as well as their dynamics. [5][6][7] By exploiting the element specificity of inner-shell excitation, X-ray absorption spectroscopy (XAS) is an ideal tool to study the local electronic and geometric structure in the vicinity of a specific element within a molecule.…”

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].…”