Multiple Electron Ejection from Proteins Resulting from Single-Photon Excitation in the Valence Shell

One-photon multiple ionization is a signature of dynamical electron correlations in atoms and small molecules, as observed in the Auger process when Auger electron emission follows core–shell ionization. In such a process, the high energy needed to remove several electrons is due to the strong Coulombic attraction between the last departing electron(s) and the ionic core. Multiply negatively charged molecules offer the possibility to overcome the Coulombic attraction, opening the way for multielectron photodetachment following valence shell excitation. Here photodetachment studies have been performed on electrosprayed protein polyanions using vacuum ultraviolet synchrotron radiation coupled to a radiofrequency ion trap. Double, triple, and quadruple electron emissions from protein polyanions resulting from single-photon excitation in the valence shell were observed with ionization thresholds below 20 eV photon energy. This suggests the existence of large electronic correlations in proteins between weakly bound electrons standing on distant sites. Besides, the resulting multiradical polyanions appear to be remarkably stable, an important issue in radiobiology

The Gas-Phase Photophysics of Eosin Y and its Maleimide Conjugate

The use of the xanthene family of dyes as fluorescent probes in a wide range of applications has provided impetus for the studying of their photophysical properties. In particular, recent advances in gas-phase techniques such as FRET that utilize such chromophores have placed a greater importance on the characterization of these properties in the gas phase. Additionally, the use of synthetic linker chains to graft the chromophores in a site-specific manner to their target system is ubiquitous. There is, however, often limited information on how the addition of such a linker chain may affect the photophysical properties of the chromophores, which is of fundamental importance for interpretation of experimental data reliant on grafted chromophores. Here, we present data on the optical spectroscopy of different protonation states of Eosin Y, a fluorescein derivative. We compare the photophysics of Eosin Y to its maleimide conjugate, and to the thioether product of the reaction of this conjugate with cysteamine. Comparison of the mass spectra following laser irradiation shows that very different relaxation takes place upon addition of the maleimide moiety but that the photophysics of the bare chromophore are restored upon addition of cysteamine. This radical change in the photophysics is interpreted in terms of charge-transfer states, whose energy relative to the S₁ ← S₀ transition of the chromophore is dependent on the conjugation of the maleimide moiety. We also show that the shape of the absorption band is unchanged in the gas-phase as compared to the solution-phase, showing a maximum with a shoulder toward the blue, and examination of isotope distributions of the isolated ions show that this shoulder cannot be due to the presence of dimers. Consideration of the fluorescence emission spectrum allows a tentative assignment of the shoulder to be due to a vibrational progression with a high Franck–Condon factor

Visible and Ultraviolet Spectroscopy of Gas Phase Rhodamine 575 Cations

The visible and ultraviolet spectroscopy of gas phase rhodamine 575 cations has been studied experimentally by action-spectroscopy in a modified linear ion trap between 220 and 590 nm and by time-dependent density functional theory (TDDFT) calculations. Three bands are observed that can be assigned to the electronic transitions S₀ → S₁, S₀ → S₃, and S₀ → (S₈,S₉) according to the theoretical prediction. While the agreement between theory and experiment is excellent for the S₃ and S₈/S₉ transitions, a large shift in the value of the calculated S₁ transition energy is observed. A theoretical analysis of thermochromism, potential vibronic effects, and–qualitatively–electron correlation revealed it is mainly the latter that is responsible for the failure of TDDFT to accurately reproduce the S₁ transition energy, and that a significant thermochromic shift is also present. Finally, we investigated the nature of the excited states by analyzing the excitations and discussed their different fragmentation behavior. We hypothesize that different contributions of local versus charge transfer excitations are responsible for 1-photon versus 2-photon fragmentation observed experimentally

Action-FRET: Probing the Molecular Conformation of Mass-Selected Gas-Phase Peptides with Förster Resonance Energy Transfer Detected by Acceptor-Specific Fragmentation

The use of Förster resonance energy transfer (FRET) as a probe of the structure of biological molecules through fluorescence measurements in solution is well-attested. The transposition of this technique to the gas phase is appealing since it opens the perspective of combining the structural accuracy of FRET with the specificity and selectivity of mass spectrometry (MS). Here, we report FRET results on gas-phase polyalanine ions obtained by measuring FRET efficiency through specific photofragmentation rather than fluorescence. The structural sensitivity of the method was tested using commercially available chromophores (QSY 7 and carboxyrhodamine 575) grafted on a series of small, alanine-based peptides of differing sizes. The photofragmentation of these systems was investigated through action spectroscopy, and their conformations were probed using ion mobility spectrometry (IMS) and Monte Carlo minimization (MCM) simulations. We show that specific excitation of the donor chromophore results in the observation of fragments that are specific to the electronic excitation of the acceptor chromophore. This shows that energy transfer took place between the two chromophores and hence that the action-FRET technique can be used as a new and sensitive probe of the structure of gas-phase biomolecules, which opens perspectives as a new tool in structural biology