International Journal of Mass Spectrometry 367 (2014) 21–27 Contents lists available at ScienceDirect International Journal of Mass Spectrometry jou rn al h om epage: www.elsevier.com/locate/ijms Ion source parameters and hydrogen scrambling in the ECD of selectively deuterated peptides Magalie Duchateau a,1 , Thomas J.D. Jørgensen b , Ophélie Robine a , Edith Nicol a , Christian Malosse a,1 , Julia Chamot-Rooke a,1 , Guillaume van der Rest a,∗ a Laboratoire des Mécanismes Réactionnels, Département de Chimie, Ecole Polytechnique and CNRS, 91128 Palaiseau Cedex, France b Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark a r t i c l e i n f o Article history: Received 24 April 2013 Received in revised form 9 April 2014 Accepted 22 April 2014 Available online 2 May 2014 Keywords: Hydrogen/deuterium exchange Electron capture dissociation Fourier transform-ion cyclotron resonance mass spectrometry Labeled peptide Electrospray ionization a b s t r a c t Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study protein dynamics in solution. Recently, electron-based fragmentation methods (ECD and ETD) have been utilized in HDX-MS/MS experiments as experimental tools to increase the spatial resolution (the ability to obtain deuterium levels of individual residues). An essential prerequisite for this approach is that the level of hydrogen scrambling is negligible. The occurrence of hydrogen scram- bling depends critically on the extent of vibrational excitation in the mass spectrometer. In particular, the desolvation process in the electrospray ion source is likely to induce scrambling at standard operat- ing conditions. Consequently, finding experimental conditions that minimize hydrogen scrambling to a negligible level is thus pivotal for the application of electron-based fragmentation in HDX-MS/MS exper- iments. In the present work, we investigate the occurrence of scrambling in the Apollo I electrospray ion source using ECD of selectively deuterium labeled peptides. The electrospray ion source settings leading to minimal scrambling were identified. Furthermore, an energy dependent loss of deuterium occurring in the ion source was also observed. This loss was critically dependent on the occurrence of scrambling. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Amide hydrogen/deuterium exchange monitored by mass spec- trometry (HDX-MS) has emerged as an important method to obtain information on the dynamics of proteins and protein complexes in solution [1–6]. Modern mass spectrometers can measure proteins at submicromolar concentrations and this allows investigations of, e.g., amyloidogenic proteins which aggregate at higher con- centrations such as those typically used for NMR studies [7]. HDX-MS experiments are particularly suited to obtain informa- tion on changes in protein structure and dynamics, for example, upon complex formation or by a change in the environment (ionic strength, temperature, solvent etc.). In HDX-MS, the standard pro- tocol relies on a proteolytic digestion of the deuterated protein followed by mass measurement of the peptide fragments, which ∗ Corresponding author. Present address: Laboratoire de Chimie Physique, Uni- versité Paris Sud, 91405 Orsay Cedex, France. Tel.: +33 1 69 15 30 41. E-mail addresses: guillaume.van-der-rest@u-psud.fr, gvdr@dcmr.polytechnique.fr (G. van der Rest). 1 Present address: Unité de Spectrométrie de Masse Structurale et Protéomique, Institut Pasteur, CNRS UMR 3528, 28 rue du Docteur Roux, 75015 Paris, France. is usually done in an ESI(-LC)-MS strategy [8], but can also be done by MALDI-MS [9–11]. The ability to localize the individual deuter- ated residues (the spatial resolution) is limited by the size of the proteolytic fragments that are obtained. Due to the experimen- tal conditions used to quench the H/D exchange of the backbone amides (low pH, low temperature), the enzyme mostly used is pepsin [12], although strategies using a combination of alternative enzymes have also been utilized [13,14]. Pepsin often yields over- lapping peptide sequences which can then be used to sublocalize the deuterium incorporation. The use of a combination of enzymes, providing additional cleavage sites, can also be used to generate an even higher number of overlapping peptides, improving the spatial resolution. Nevertheless, these enzymatic strategies do not usually result in single-residue resolution, except for a few residues. An alternative strategy is to follow what has become standard in proteomics, i.e. resort to an MS/MS approach, fragmenting the pep- tides to obtain not their sequence as is traditional in proteomics, but to calculate the amount of deuterium present on the various fragment ions. In order for this approach to be worthwhile, one major requirement is that the fragmentation process in itself should not change the initial position of the amide deuterium or hydro- gen atoms. Otherwise, the initial deuteration pattern present in http://dx.doi.org/10.1016/j.ijms.2014.04.007 1387-3806/© 2014 Elsevier B.V. All rights reserved.