The low density of ions in mass spectrometers generally precludes direct infrared (IR) absorption measurements. The IR spectrum of an ion can nonetheless be obtained by inducing photodissociation of the ion using a high-intensity tunable laser. The emergence of free electron lasers (FELs) and recent breakthroughs in bench-top lasers based on nonlinear optics have now made it possible to routinely record IR spectra of gas-phase ions. As the energy of one IR photon is insufficient to cause dissociation of molecules and strongly bound complexes, two main experimental strategies have been developed to effect photodissociation. In infrared multiple-photon dissociation (IR-MPD) many photons are absorbed resonantly and their energy is stored in the bath of vibrational modes, leading to dissociation. In the "messenger" technique a weakly bound van der Waals atom is detached upon absorption of a single photon. Fundamental, historical, and practical aspects of these methods will be presented. Both of these approaches make use of very different methods of ion preparation and manipulation. While in IR-MPD ions are irradiated in trapping mass spectrometers, the "messenger" technique is generally carried out in molecular beam instruments. The main focus of this review is the application of IR spectroscopy to biologically relevant molecular systems (amino acids, peptides, proteins). Particular issues that will be addressed here include gas-phase zwitterions, the (chemical) structures of peptides and their collision-induced dissociation (CID) products, IR spectra of gas-phase proteins, and the chelation of metal-ligand complexes. Another growing area of research is IR spectroscopy on solvated clusters, which offer a bridge between the gas-phase and solution environments. The development of state-of-the-art computational approaches has gone hand-in-hand with advances in experimental techniques. The main advantage of gas-phase cluster research, as opposed to condensed-phase experiments, is that the systems of interest can be understood in detail and structural effects can be studied in isolation. It will be shown that IR spectroscopy of mass-selected (bio)molecular systems is now well-placed to address specific questions on the individual effect of charge carriers (protons and metal ions), as well as solvent molecules on the overall structure.

The gas-phase structures of the protonated pentapeptide Leu-enkephalin and its main collision-induced dissociation (CID) product ions, b4 and a4, are investigated by means of infrared multiple-photon dissociation (IR-MPD) spectroscopy and detailed molecular mechanics and density functional theory (DFT) calculations. Our combined experimental and theoretical approach allows accurate structural probing of the site of protonation and the rearrangement reactions that have taken place in CID. It is shown that the singly protonated Leu-enkephalin precursor is protonated on the N-terminus. The b4 fragment ion forms two types of structures: linear isomers with a C-terminal oxazolone ring, as well as cyclic peptide structures. For the former structure, two sites of proton attachment are observed, on the N-terminus and on the oxazolone ring nitrogen, as shown in a previous communication (Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2005, 127, 17154-17155). Upon leaving the ions for longer radiative cooling delays in the ion cyclotron resonance (ICR) cell prior to IR spectroscopic investigation, one observes a gradual decrease in the relative population of oxazolone-protonated b4 and a corresponding increase in N-terminal-protonated b4. This experimentally demonstrates that the mobile proton is transferred between two sites in a gas-phase peptide ion and allows one to rationalize how the proton moves around the molecule in the dissociation process. The a4 fragment, which is predominantly formed via b4, is also confirmed to adopt two types of structures: linear imine-type structures, and cyclic structures; the former isomers are exclusively protonated on the N-terminus in sharp contrast to b4, where a mixture of protonation sites was found. The presence of cyclic b4 and a4 fragment ions is the first direct experimental proof that fully cyclic structures are formed in CID. These results suggest that their presence is significant, thus lending strong support to the recently discovered peptide fragmentation pathways (Harrison, A. G.; Young, A. B.; Bleiholder, B.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2006, 128, 10364-10365) that result in scrambling of the amino acid sequence upon CID.

Infrared multiple-photon dissociation (IR-MPD) spectroscopy has been applied to singly-charged complexes involving the transition metals Ag + and Zn 2+ with the aromatic amino acid phenylalanine. These studies are complemented by DFT calculations. For [Phe+Ag] + the calculations favor a tridentate charge solvation N/O/ring structure. The experimental spectrum strongly supports this as the predominant binding geometry and, in particular, rules out a significant presence of the salt-bridge conformation. Zn 2+ forms a deprotonated dimer complex with Phe, [Zn+Phe2-H] + , in which the +2 oxidation state serves as a useful biomimetic model for zinc protein sites. A number of low-energy conformations were located, of which the lowest-energy conformer predicted by the calculations involves a Phe ligand deprotonated on the carboxylic acid, while the other Phe ligand is in the tridentate charge solvation conformation. The calculated IR spectrum of this conformer gives a close fit to the experimental spectrum, strongly supporting this as the predominant binding geometry. This most stable calculated complex is characterized by N/ O/ring metal chelation with a tetrahedral-type coordination core of Zn 2+ to N and O of both ligands. Another similar tightly chelated structure shows a square-planar-type coordination core, but this structure is computed to be less stable and gives a less satisfactory match to the experimental spectrum. This preference for the tetrahedral geometry of the Lewis-basic atomic ligands parallels the common Zn(II) coordination geometry in proteins. The number of clearly identifiable peaks resolved in the IR-MPD spectra as well as the much-improved matches between the observed spectra and the DFT-calculated spectra of the most stable geometries compared to previous studies are noteworthy for systems of this size and complexity. These results demonstrate that IR spectroscopy of transition metal-amino acid complexes in combination with DFT calculations is a very powerful structural tool, readily applicable to biomimetic systems that model, for example, the reaction centers of proteins in the solvent-free environment. In addition, we present a novel ion-capturing method for Fourier transform ion cyclotron resonance mass spectrometry which removes the necessity of a buffer gas pulse, while allowing ion trapping at moderate voltages with apparently reduced collisional excitation of the ions.