Coupling of HPLC with Electrospray Ionization Mass Spectrometry for Studying the Aging of Ultrasmall Multifunctional Gadolinium-Based Silica Nanoparticles

Sub-5 nm multimodal nanoparticles have great potential for theranostic applications due to their easy renal elimination combined with complementary imaging properties and therapeutic facilities. Their potential clinical use requires the full characterization of not only the nanoparticle but also all its possible degradation products. We have recently proposed new ultrasmall gadolinium-based nanoparticles for multimodal imaging and radiosensitization. The aim of this article is to describe an analytical tool to characterize degradation products in a highly diluted medium. We demonstrate that HPLC coupled to electrospray ionization mass spectrometry (ESI-MS) can be used in order to determine precisely the composition of nanoparticles and their degradation fragments during aging

Correlating Droplet Size with Temperature Changes in Electrospray Source by Optical Methods

We investigated how the temperature and size of charged droplets are affected by the electrospray ionization (ESI) process, using <i>in situ</i> measurements involving laser-induced fluorescence and Mie scattering on a thermal gradient focusing ESI source. Rhodamine dyes were employed as temperature indicators using ratiometric intensity-based fluorescence techniques. The results were compared to lifetime-based techniques using tris­(2,2′-bipyridyl)­dichlororuthenium­(II) hexahydrate, [Ru­(bpy)₃]²⁺. Both methods gave similar profiles. Nevertheless, the precision and sensitivity were higher for lifetime-based techniques in comparison to intensity-based techniques. Global warming (with Δ<i>T</i> ∼10 K) of the ESI plume is reported while the size of the droplet decreases along the plume. The global warming indicates that the conductive thermal transfer (between the superheated sheath gas and the solvent) is predominant and stronger than the cooling effect due to the evaporation of the droplets, and this outcome is effectively reproduced by a diffusion-controlled evaporation model. Thermal gradient focusing ESI sources therefore appear to be efficient sources for evaporating large amounts of solvent, along with an increase in temperature

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

UV Spectroscopy of DNA Duplex and Quadruplex Structures in the Gas Phase

UV absorption spectroscopy is one of the most widely used methods to monitor nucleic acid folding in solution, but the absorption readout is the weighted average contribution of all species present in solution. Mass spectrometry, on the other hand, is able to separate constituents of the solution based on their mass, but methods to probe the structure of each constituent are needed. Here, we explored whether gas-phase UV spectroscopy can give an indication of DNA folding in ions isolated by electrospray mass spectrometry. Model DNA single strands, duplexes, and G-quadruplexes were extracted from solution by electrospray; the anions were stored in a quadrupole ion trap and irradiated by a tunable laser to obtain the UV action spectra of each complex. We found that the duplex and quadruplex spectra are significantly different from the spectra of single strands, thereby suggesting that electronic spectroscopy can be used to probe the DNA gas-phase structure and obtain information about the intrinsic properties of high-order DNA structure

Correlation between the Charge of Polymer Particles in Solution and in the Gas Phase Investigated by Zeta-Potential Measurements and Electrospray Ionization Mass Spectrometry.

The relationship between the effective charge of polymer nanoparticles (PNP) in solution and the charge states of ionized particles produced in the gas phase by electrospray ionization was investigated. Charge detection mass spectrometry was used to measure both the mass and charge of individual electrosprayed ions. The effective charges extracted from the measured zeta-potential of PNPs in solution are partially correlated with the average values of charge of PNPs in the gas phase. The correlation between the magnitude of charging of PNPs ions produced in the gas phase with the PNPs surface charge in solution demonstrates that the mass spectrometry-based analysis described in this work is an alternative and promising way for a fast and systematic characterization of charges on colloidal particles

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

Hydrogen-Induced Adsorption of Carbon Monoxide on the Gold Dimer Cation: A Joint Experimental and DFT Investigation

It is demonstrated, using tandem mass spectrometry and radio frequency ion trap, that the adsorption of a H atom on the gold dimer cation, Au₂H⁺, prevents its dissociation and allows for adsorption of CO. Reaction kinetics are measured by employing a radio frequency ion trap, where Au₂⁺ and CO interact for a given reaction time. The effect of a hydrogen atom is evaluated by comparing reaction rate constants measured for Au₂⁺ and Au₂H⁺. The theoretical results for the adsorption of CO molecules and their reaction characteristics with Au₂⁺ and Au₂H⁺ are found to agree with the experimental findings. The joint investigations provide insights into hydrogen atom adsorption effects and consequent reaction mechanisms

Direct Molar Mass Determination of Self-Assembled Amphiphilic Block Copolymer Nanoobjects Using Electrospray-Charge Detection Mass Spectrometry

Charge detection mass spectrometry (CD-MS) combined with electrospray ionization was used to determine, in a direct way and for the first time, the molar mass of self-assembled amphiphilic block copolymer nanoobjects prepared via living radical emulsion polymerization. CD-MS supplies enough data for calculating statistically significant measurements of the mass of particles in the megadalton to gigadalton range and their resulting mass distribution

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