Infrared photofragment spectroscopy of charged amino acid water clusters in the gas phase

We present in this work a new technique, which combines laser photofragmentation spectroscopy with tandem mass spectrometry, for structural investigations of biomolecular ions in the gas phase. A novel apparatus was designed and built for the implementation of these studies. In this instrument closed shell ions are produced in the gas phase by electrospray ionization and stored in a hexapole ion trap prior to mass selection of a parent beam in a first quadrupole and then irradiation in an octupole ion guide using laser light. Mass analysis of the photofragmentaion products in a final stage quadrupole as a function of the wavelength generates an action spectrum. We applied this new technique to follow the microsolvation of charged amino acids in the gas phase. In these studies, the nanoelectrospray ionization source generates a distribution of water clusters of charged amino acids at various hydration levels. A particular size of cluster is selected and irradiated by infrared laser pulses resulting in the dissociation of one water molecule. Detection of the photofragmentation of this weak non-covalent bond allows us to generate the vibrational action spectrum of this particular cluster. We use a two-stage difference frequency mixing setup to produce laser light in the 2900–3800 cm-1, allowing us to probe the light-atom stretching region. IR photofragmentation spectra have been obtained for the hydrates of protonated and lithiated valine and those of protonated tryptophan. By probing the region of free and hydrogen-bonded N-H and O-H stretch vibrations and with the help of density functional theory calculations at the B3LYP/6-31++G** level, we relate spectral changes to the structure of clusters. In the study of lithiated valine water clusters, we addressed the question of zwitterion formation upon the combined effect of water and an external ion. Our data indicate the presence of the non-zwitterionic form of valine upon addition of up to four water molecules. In all of the species studied here, the hydration process is driven by solvation of the charge, and upon completion of a first shell around it, water preferentially forms a second solvation shell with no strong competition from other hydration sites on the amino acid backbone. Strikingly, similar water network structures have been observed at the highest hydration level of completely different species, probably indicating the existence of stable ordered water structures. We obtained evidence for a structrual change of the valine backbone in lithiated valine upon addition of the third water molecule, while no conformational change has been identified in the clusters of the protonated species. We have thus been able to answer questions related to the conformational preferences of the amino acid and the structuring of the water network.

Spectroscopy and dissociation dynamics of cold, biomolecular ions

Monia Guidi

The function of a peptide is intimately connected to its structure, which in solution is the result of the balance between non-covalent interactions within the molecule and the molecule-solvent interactions. Theory has the power to predict the properties of a wide variety of molecules, but the accuracy of the theoretical predictions must be verified by experiments. A way to test, and hopefully improve, theory is to study proteins or peptides in the gas-phase. The combination of mass spectrometry and laser spectroscopy is used to investigate properties of biological ions in the gas-phase. Closed-shell biomolecular ions produced by an electrospray source are cooled down in an RF ion trap to less than 10 K and interrogated spectroscopically. Electronic and conformer-specific vibrational spectra have been measured for small protonated peptides applying photofragment-based detection schemes. These measurements allow us to investigate the role of the charge and the influence of the nature and the position of the chromophore in the peptide chain on its conformational preferences. Highly-resolved conformer-specific vibrational spectra of such species, as well as those of a double-chromophore molecule, help elucidate the mechanism of the IR-UV double-resonance methods and provide benchmarks for testing the accuracy of theoretical calculations. This thesis shows also the experimental developments that allow us to use photofragment-based detection scheme to measure highly-resolved electronic spectra of peptides of up to 17 amino acids, containing one or two chromophores, using infrared laser-assisted photofragment spectroscopy (IRLAPS). The fragmentation yield of UV pre-excited molecules increases by as much as two orders of magnitude when they further interact with the CO2 laser. This approach can be also applied in a IR-UV double resonance scheme, allowing measurements of conformer-specific infrared spectra of protonated peptides. The fragmentation channel, which is always greatly enhanced by IRMPE of UV pre-excited molecules, is the loss of the neutral, aromatic side chain via cleavage of the Cα-Cβ bond, independent of the chromophore excited in the peptide. The possible mechanisms, involving the formation of a long-lived intermediate species, that lead to the formation of the observed photofragment are discussed in this thesis work.

EPFL2010

Electronic and vibrational spectroscopy of cold protonated amino acids in the gas phase

Sébastien Mercier

In this thesis report, we describe a novel home-built instrument designed to study the spectroscopy of biomolecular ions in the gas phase and at low temperature, and we present the first experimental results obtained on protonated aromatic amino acids. The apparatus consists in a tandem mass spectrometer equipped with a nanospray ion source and a cryocooled 22-pole ion trap. The charged species produced by the source traverse a first quadrupole mass filter and mass-selected ions are then accumulated and thermalized in the ion trap, where their vibrational temperature can be lowered to ~ 10 K. Two setups allow us to generate ultraviolet and infrared laser light to spectroscopically probe the trapped ions by photodissociation. The resulting fragment ions are finally released from the trap and mass analyzed by a second quadrupole spectrometer. Photodissociation electronic spectra have been measured for the protonated amino acids tryptophan (TrpH+) and tyrosine (TyrH+), as well as for hydrated complexes of the former. The spectrum of TyrH+ exhibits sharp, fully resolved vibronic transitions, which attests to the low temperature of the ions. In contrast, that of tryptophan shows a broad absorption band that covers several hundreds of wavenumbers. This has been attributed to lifetime broadening related to an ultrafast (< ~ 100 fs) deactivation mechanism of the TrpH+ S1(ππ*) excited state. This phenomenon is caused by a strong coupling between this ππ* state and a nearby dissociative state of πσ* character mainly localized on the charged ammonium group. Solvation of TrpH+ by only two water molecules is sufficient to significantly destabilize the σ* orbitals and reduce that coupling, which results in a longer excited-state lifetime and a fully resolved electronic spectrum. These interpretations are supported by ab initio and density functional theory (DFT) calculations. Infrared-ultraviolet double resonance spectroscopic techniques have been successfully implemented to identify the different conformers of TyrH+ and doubly hydrated protonated tryptophan (TrpH+·W2) by measuring conformer-specific vibrational and electronic spectra for each of them. By comparison with the results of DFT geometry and harmonic frequency calculations, we could confidently determine the structures of the TyrH+ conformers and suggest possible assignments for those of TrpH+·W2. Two important stabilizing interactions could be inferred from these results: (i) the attraction between the charged ammonium group and the π-electron cloud of the aromatic ring; (ii) the hydrogen-bonding interaction of a donor water molecule to the acceptor indole ring of TrpH+·W2.

EPFL2008

Infrared spectroscopy of hydrated amino acids in the gas phase: protonated and lithiated valine

Rainer Beck, Sébastien Mercier, Thomas Rizzo, Evan Rowland Williams

We report here infrared spectra of protonated and lithiated valine with varying degrees of hydration in the gas phase and interpret them with the help of DFT calculations at the B3LYP/6-31++G** level. In both the protonated and lithiated species, our results clearly indicate that the solvation process is first driven by solvation of the charge site and subsequently by formation of a second solvation shell. The infrared spectra of Val•Li+(H2O)4 and Val•H+(H2O)4 are strikingly similar in the region of the spectrum corresponding to hydrogen-bonded stretches of donor water molecules, suggesting that in both cases similar extended water structures are formed once the charge site is solvated. In the case of the lithiated species, our spectra are consistent with a conformation change of the amino acid backbone from the syn to anti accompanied by a change in the lithium binding from NO coordination to OO coordination configuration upon addition of the third water molecule. This change in the mode of metal ion binding was also observed previously by Williams and Lemoff [J. Am. Soc. Mass Spectrom. 2004, 15, 1014-1024] using blackbody infrared radiative dissociation (BIRD). In contrast to the zwitterion formation inferred from results of the BIRD experiments upon the addition of a third water molecule, our spectra, which are a more direct probe of structure, show no evidence for zwitterion formation with the addition of up to four water molecules.