Spectroscopic studies of cold, gas-phase biomolecular ions

While the marriage of mass spectrometry and laser spectroscopy is not new, developments over the last few years in this relationship have opened up new horizons for the spectroscopic study of biological molecules. The combination of electrospray ionization for producing large biological molecules in the gas phase together with cooled ion traps and multiple- resonance laser schemes are allowing spectroscopic investigation of individual conformations of peptides with more than a dozen amino acids. Highly resolved infrared spectra of single conformations of such species provide important benchmarks for testing the accuracy of theoretical calculations. This review presents a number of techniques employed in our laboratory and in others for measuring the spectroscopy of cold, gas-phase protonated peptides. We show examples that demonstrate the power of these techniques and evaluate their extension to still larger biological molecules.

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

Spectroscopy of mobility-selected biomolecular ions

Georgios Papadopoulos, Thomas Rizzo, Annette Svendsen

We describe here experiments that combine differential ion mobility, which separates conformational isomers of biomolecular ions, with electronic spectroscopy in a cold, radio-frequency ion trap. Although the low temperature attainable in a cold ion trap greatly simplifies the electronic spectra of large molecules, conformational heterogeneity can still be a significant source of congestion, complicating spectroscopic analysis. We demonstrate here that using differential ion mobility to separate gas-phase peptide conformers before injecting them into a cold ion trap allows one to decompose a dense spectrum into contributions from different conformational families. In the inverse sense, cold ion spectroscopy can be used as a conformation-specific detector for ion mobility, allowing one to separate an unresolved peak into contributions from different conformational families. The doubly protonated peptide bradykinin serves as a good test case for the marriage of these two techiques as it exhibits a considerable degree of conformational heterogeneity that results in a highly congested electronic spectrum. Our results demonstrate the feasiblity and advantages of directly coupling ion mobility with spectroscopy and provide a diagnostic of conformational isomerization of this peptide after being produced in the gas phase by electrospray.

2011

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.