International Journal of Mass Spectrometry 297 (2010) 162–169 Contents lists available at ScienceDirect International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms Hydrogen/deuterium exchange of phenylalanine analogs studied with infrared multiple photon dissociation Cesar S. Contreras a,1 , Nicolas C. Polfer a , Alfred C. Chung b , Jos Oomens c , John R. Eyler a,∗ a Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611-7200, USA b Interdisciplinary Center for Biotechnology Research, Proteomics Division, University of Florida, Gainesville, FL, USA c FOM-Institute for Plasma Physics Rijnhuizen, Nieuwegein, The Netherlands article info Article history: Received 11 May 2010 Received in revised form 18 August 2010 Accepted 19 August 2010 Available online 6 September 2010 Keywords: FTICR Amino acids IRMPD Ab initio Hydrogen–deuterium exchange Phenylalanine abstract Phenylalanine analogs were subjected to hydrogen/deuterium exchange (HDX) in both solution and the gas phase, and gas-phase infrared multiple photon dissociation spectra were obtained for each of the species. For sodium cation-attached N-acetylphenylalanine, gas-phase HDX took place at only one site. Comparison of spectra from both undeuterated and singly deuterated sodiated N-acetylphenylalanine showed band shifts for normal modes that involved mainly vibrations of the O–H group, indicating that gas-phase exchange occurs at the COOH hydrogen and not at the NH hydrogen. Conversely, HDX in solution did result in exchange of the NH hydrogen, even for the protected species O-methyl N- acetylphenylalanine and N-acetylphenylalanine O-methylglycine. Rate coefficients for gas-phase H/D exchange were measured for the single deuteration of sodiated N-acetylphenylalanine and all three deuterations of protonated N-acetylphenylalanine, and found to be in the range (1.5–3.6) × 10 -11 cm 3 /s. Density functional theory calculations predicted that the phenylalanine analogs, although of different size, have relatively similar structural features. These calculations showed that Na + interacts with the phenyl ring and all available carbonyl oxygens, thus essentially locking the structures into one basic conformation. This behavior is quite distinct from other amino acids which are more flexible, and where gas-phase exchange also occurs at the amine (NH) group. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Hydrogen deuterium exchange (HDX) is a method in which par- ticularly labile hydrogen atoms are substituted with deuterium atoms in a molecule or ion of interest [1–8]. H/D exchange has been used in solution-phase studies to obtain structural information for large biomolecules using both mass spectrometric [5,8,9] and nuclear magnetic resonance (NMR) [10] techniques. H/D exchange studies have also been conducted in the gas phase and early mass spectrometric work from Beauchamp et al. probed the pro- ton affinity and exchange rates of labile hydrogen atoms [11], while ion–molecule reactions involving H/D exchange have also been followed in mass spectrometry experiments [12]. Utilizing the technique of infrared multiple photon dissociation (IRMPD) [13–18], infrared spectra of gas-phase deuterated ions can be obtained, and these have recently been used to obtain structural information for small ions [19–27] and biologically relevant ions ∗ Corresponding author. Tel.: +1 352 392 0532; fax: +1 352 392 0872. E-mail address: eylerjr@chem.ufl.edu (J.R. Eyler). 1 Present address: NASA Ames Research Center, Moffett Field, CA, USA. [28–30], occasionally observing the spectral shifts that occur in the infrared spectra due to deuteration [31]. H/D exchange experiments can provide a great deal of structural information, especially when coupled with mass spectrometric and/or infrared spectral techniques. Substitution of hydrogen by deuterium has primarily been used to obtain information on the tertiary structure of proteins [9], but can also provide primary struc- tural information for smaller species such as the phenylalanine analogs studied in this work. In the case of amino acids, exchange is seen for hydrogen atoms bound to the Lewis base oxygen and nitrogen atoms [32]. Phenylalanine (Phe), one of the essential amino acids, has been studied extensively both in solution and in the gas phase [30,33–39]. Recent work has shown that Phe behaves differently in each phase, in part due to structural differences of the molecule in the two phases [40]. The binding of alkali metals to Phe in the gas phase has been especially well characterized [35–37]. How- ever, much less is known of the effect that protecting groups or modifications to the amino acid have on the phenylalanine struc- ture. Recent work by Dunbar and co-workers [30] showed that when the backbone is extended, e.g., by forming a dipeptide, an attached alkali cation has a higher possibility of forming a chelating 1387-3806/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijms.2010.08.019