Determination of Peptide Topology through Time-Resolved Double- Resonance under Electron Capture Dissociation Conditions Marie Pe ́ rot-Taillandier, †,‡ Se ́ verine Zirah, † Sylvie Rebuffat, † Uwe Linne, § Mohamed A. Marahiel, § Richard B. Cole, ‡ Jean-Claude Tabet, ‡ and Carlos Afonso* ,‡,⊥ † National Museum of Natural History, Communication Molecules and Adaptation of Microorganisms, UMR 7245 CNRS - MNHN, CP 54, 57 rue Cuvier, F-75005 Paris, France ‡ University Pierre & Marie Curie, Parisian Institute of Molecular Chemistry, UMR 7201 CNRS - UPMC, 4 place Jussieu, F-75005 Paris, France § Philipps-University Marburg, Department of Chemistry, Fb. 15-Chemie, Hans-Meerwein-Straße, D-35032 Marburg, Germany ⊥ University of Rouen, UMR 6014 CNRS COBRA, INSA of Rouen, 1 rue Tesnie ̀ re, 76821 Mont Saint-Aignan, France * S Supporting Information ABSTRACT: Characterizing the conformation of biomolecules by mass spectrometry still represents a challenge. With their knotted structure involving a N-terminal macrolactam ring where the C-terminal tail of the peptide is threaded and sterically trapped, lasso peptides constitute an attractive model for developing methods for characterizing gas-phase conformation, through comparison with their unknotted topoisomers. Here, the kinetics of electron capture dissociation (ECD) of a lasso peptide, capistruin, was investigated by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry and compared to that of its branched-cyclic topoisomer, lactam- capistruin. Both peptides produced rather similar ECD spectra but showed different extent of H • transfer from c i ˈ to z j • ions. Time-resolved double-resonance experiments under ECD conditions were performed to measure the formation rate constants of typical product ions. Such experiments showed that certain product ions, in particular those related to H • transfer, proceeded through long-lived complexes for capistruin, while fast dissociation processes predominated for lactam-capistruin. The formation rate constants of specific ECD product ions enabled a clear differentiation of the lasso and branched-cyclic topoisomers. These results indicate that the formation kinetics of ECD product ions constitute a new way to explore the conformation of biomolecules and distinguish between topoisomers and, more generally, conformers. I n recent years, the use of electrospray ionization mass spectrometry (ESI-MS) to characterize the gas-phase conformation of biomolecules has grown considerably. Differ- ent methods have emerged, from the analysis of charge state distributions 1 and H/D exchange profiles 2 to the interpretation of intramolecular cross-links 3 or ion mobility profiles. 4 Dissociation profiles can also be informative on the gas-phase conformation of biomolecules, in particular electron capture dissociation (ECD). 5-8 For peptides and proteins, ECD generates mainly complementary c i ˈ and z j • product ions from dissociation of the charge-reduced molecular ion. 5,9 ECD has found success at investigating the location of noncovalent interactions, 6,10,11 since it can trigger dissociations of positively charged complexes without significantly perturbing the non- covalent interactions. This feature can be generalized to the intramolecular interactions governing conformations. 12 As an example, an attempt to rationalize the periodicity in the relative abundance of ECD cˈ/z • product ions has been performed by Tsybin and colleagues for peptides for which an α helix conformation was maintained in the gas phase. 13 In addition, ECD can be accompanied by intramolecular H • transfers (from c i ˈ to z j • complementary product ions in peptides and proteins). 14-18 This process is sequence dependent and relies on the gas-phase basicity properties as well as the proximity of the interacting groups. 14,17 It has been proposed to take place within long-lived [c i ˈ,z j • ] ion-dipole or ion-ion complexes, 15 therefore suggesting that it could be strongly influenced by the gas-phase conformation. 15,18-21 An attractive method to understand the kinetics governing ECD and the associated H • processes is provided by double- resonance (DR) experiments, based on the resonant excitation of an intermediate ion. DR has been developed in the 1960s to highlight intermediate ions produced by consecutive processes into the drift cell of an ICR mass spectrometer and has been rapidly applied to the characterization of ion-molecule reaction kinetics. 22 More recently, this methodology has been successfully used in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), using different activation processes: ECD, 20 electron-detachment dissociation (EDD), 23-25 and infrared multiple photon dissociation Received: March 1, 2012 Accepted: May 4, 2012 Published: May 4, 2012 Article pubs.acs.org/ac © 2012 American Chemical Society 4957 dx.doi.org/10.1021/ac300607y | Anal. Chem. 2012, 84, 4957-4964