Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

Hisham Ben Hamidane 0 1 4 Diego Chiappe 0 1 2 Ralf Hartmer 0 1 3 Aleksey Vorobyev 0 1 4 Marc Moniatte 0 1 2 Yury O. Tsybin 0 1 4 0 Address reprint requests to Prof. Yury O. Tsybin, Ecole Polytechnique Fdrale de Lausanne, Biomolecular Mass Spectrometry Laboratory , BCH 4307, 1015 Lausanne, Switzerland 1 Published online November 27, 2008 Received May 7, 2008 Revised October 24, 2008 Accepted November 20, 2008 2 Proteomics Core Facility, Ecole Polytechnique Fdrale de Lausanne, Lausanne, Switzerland 3 Bruker Daltonics GmbH, Bremen, Germany 4 Biomolecular Mass Spectrometry Laboratory , Ecole Polytechnique Fdrale de Lausanne, Lausanne, Switzerland We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD. (J Am Soc Mass Spectrom 2009, 20, 567-575) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry - attributed to radical intermediates ejection from the ICR trap immediately upon formation [20, 21]. An alternative approach to DR-ECD is to compare ECD fragmentation patterns obtained at room-temperature (300 K) and cold (86 K) ICR ion trap conditions [22]. In cold ICR trap long-lived radical intermediates remain inside of the trap but do not have sufficient internal energy to initiate product ion separation and thus do not contribute to the product ion mass spectrum [9, 22]. In general, long-lived radical intermediates exhibit a higher yield of radical N-terminal product ions, c ions, and even-electron or prime C-terminal product ions, z= ions than that of short-lived intermediates, presumably as the result of increased probability of hydrogen atom transfer between ECD products [9, 23]. Ion internal energy variation in activated ion (AI)-ECD [24] was shown to influence hydrogen atom rearrangement between ECD products and determine the ratio of radical to prime product ions [9, 21]. Consideration of hydrogen atom loss/gain is important for correct product ion assignment and error-free peptide sequencing in proteomics [23, 25]. Implementation of electron-transfer dissociation (ETD) in ion trap mass spectrometry further catalyzed application of electron-induced fragmentation reactions in peptide and protein sequencing and post-translational modification characterization [26 30]. Compared with ECD Dproved peptide and protein structural analysis evelopment of recent analytical methods for imhas been directed by a combination of complementary tandem mass spectrometry (MS/MS) methods [13]. Particular advances have been achieved as a result of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) based electron capture dissociation (ECD) [4] complementarity to slow heating fragmentation methods [5], such as collision-induced dissociation (CID) [6] and infrared multiphoton dissociation (IRMPD) [7]. In addition to mainly product ion mass-based MS/ MS, product ion abundance (PIA) in ECD is increasingly considered as a new source of information to improve peptide and protein sequencing [8, 9], quantitative modification analysis [10, 11], higher-order structure characterization [8, 1215], providing new insights into ECD mechanism [16, 17], suggesting charge location in peptides and proteins [18, 19], and indicating routes toward developing a quantitative model of ECD/ETD [15]. Double-resonance (DR) ECD, with and without ion preactivation, is used to estimate the radical intermediate lifetimes and differentiate between short-lived and long-lived intermediates by monitoring PIA variation FT-ICR MS of doubly charged peptides, ETD in ion trap MS typically demonstrates more abundant chargereduced radical intermediates and less extensive fragmentation pattern, indicating lower ion internal energy in ion trap-based ETD than that during ECD in an ICR ion trap [31]. Additional ion activation, or collision-induced dissociation of charge-reduced species (CRCID) [32] enhances PIA in ETD to a substantially higher degree than ion activation in ECD, especially for doubly charged precursor ions. The fragmentation pattern of ETD CRCID performed in ion trap MS seems to correlate with ECD in FT-ICR MS, whereas ETD without CRCID correlates with ECD in a low-vacuum quadrupole ion trap [33]. Indeed, it is believed nowadays that ECD and ETD produce similar fragmentation patterns. Are ECD and ETD truly similar? What method in low-vacuum ETD can be alternative or complementary to the high-vacuum ECD-based methods of peptide and protein structure analysis, such as DRECD? Here, we first present an ETD-based method of distinguishing radical intermediates by their lifetimes as a complement to double-resonance ECD. In the following, we demonstrate the distinct differences in radical/prime PIA ratio between ECD and ETD. We rationalize the observed dependencies as a function of ion internal energy. Sample Preparation Standard peptides were purchased from Sigma Aldrich (Buchs, Switzerland). Peptides LLLLALLLKO OH, SDREYPLLIROOH, and a series of HO RAAAAXAAAAKOOH peptideswhere X is one of 20 natural amino acids or a phosphorylated T, Y, or Swere produced by solid-state Fmoc chemistry on an Applied Biosystems 433A synthesizer with further purification by liquid chromatography (Protein and Peptide Synthesis Facility, Biochemistry Department, University of Lausanne, Switzerland). Peptides were dissolved in water to approximately 1 mM concentration and further diluted in a standard spraying solution (H2O/CH3OH 50:50 volume ratio with 1% HCOOH) to a final peptide concentration of about 1 M. ETD-based Tandem Mass Spectrometry ETD/CRCID experiments were performed on an ion trap mass spectrometer (HCTultra PTM discovery system, Bruker Daltonics GmbH, Bremen, Germany) by independent and subsequent application of ETD, CID, or CRCID inside the spherical ion trap [3 (...truncated)