Reaction products in mass spectrometry elucidated with infrared spectroscopy

Determining the structure and dynamics of large biologically relevant molecules is one of the key challenges facing biology. Although X-ray crystallography (XRD) and nuclear magnetic resonance (NMR) yield accurate structural information, they are of limited use when sample quantities are low. Mass spectrometry (MS) on the other hand has been very successful in analyzing biological molecules down to atto-mole quantities and has hence begun to challenge XRD and NMR as the key technology in the life sciences. This trend has been further assisted by the development of MS techniques that yield structural information on biomolecules. Of these techniques, collision-induced dissociation (CID) and hydrogen/deuterium exchange (HDX) are among the most popular. Despite advances in applying these techniques, little direct experimental evidence had been available until recently to verify their proposed underlying reaction mechanisms. The possibility to record infrared spectra of mass-selected molecular ions has opened up a novel avenue in the structural characterization of ions and their reaction products. On account of its high pulse energies and wide wavelength tunability, the free electron laser for infrared experiments (FELIX) at FOM Rijnhuizen has been shown to be ideally suited to study trapped molecular ions with infrared photo-dissociation spectroscopy. In this paper, we review recent experiments in our laboratory on the infrared spectroscopic characterization of reaction products from CID and HDX, thereby corroborating some of the reaction mechanisms that have been proposed. In particular, it is shown that CID gives rise to linear fragment ion structures which have been proposed for some time, but also yields fully cyclical ring structures. These latter structures present a possible challenge for using tandem MS in the sequencing of peptides/proteins, as they can lead to a scrambling of the amino acid sequence information. In gas-phase HDX of an amino acid it is shown that the structure can be changed from a charge solvated to a zwitterionic structure, thereby demonstrating that HDX can be an invasive technique, in fact changing the structure of the analyte. These results emphasize that more fundamental work is required in order to understand the underlying mechanisms in two of the most important structural techniques in MS.