Electron Capture Dissociation for Structural Characterization of Multiply Charged Protein Cations

Abstract

For proteins of < 20 kDa, this new radical site dissociation method cleaves different and many more backbone bonds than the conventional MS/MS methods (e.g., collisionally activated dissociation, CAD) that add energy directly to the even-electron ions. A minimum kinetic energy difference between the electron and ion maximizes capture; a 1 eV difference reduces capture by 10(3). Thus, in an FTMS ion cell with added electron trapping electrodes, capture appears to be achieved best at the boundary between the potential wells that trap the electrons and ions, now providing 80 +/- 15% precursor ion conversion efficiency. Capture cross section is dependent on the ionic charge squared (z2), minimizing the secondary dissociation of lower charge fragment ions. Electron capture is postulated to occur initially at a protonated site to release an energetic (approximately 6 eV) H. atom that is captured at a high-affinity site such as -S-S- or backbone amide to cause nonergodic (before energy randomization) dissociation. Cleavages between every pair of amino acids in mellitin (2.8 kDa) and ubiquitin (8.6 kDa) are represented in their ECD and CAD spectra, providing complete data for their de novo sequencing. Because posttranslational modifications such as carboxylation, glycosylation, and sulfation are less easily lost in ECD than in CAD, ECD assignments of their sequence positions are far more specific.

Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry

“…After being frozen in Ϫ80°C and thawed, any precipitate was resuspended in 6 M urea. (Foster City, CA) ESI/MS and MS/MS data were acquired on a 6 T modified FTMS with nano-electrospray [22][23][24][25][26]. Specific ions were isolated using stored waveform inverse Fourier-transform [32], followed by sustained off-resonance irradiation collisionally-activated dissociation (CAD) [33,34], infrared multiphoton dissociation (IRMPD) [35], and activated ion ECD [26].…”

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

“…The top-down MS strategy allows analysis of intact proteins to obtain a comprehensive assessment and localization of post-translational modifications (PTMs) and point mutations with full sequence coverage and without a priori knowledge. [27][28][29][30][31][32][33][34][35][36][37] It starts with measuring molecular weights of intact proteins followed by fragmentation of the protein in the mass spectrometer with tandem mass spectrometry (MS/MS) such as electron capture dissociation (ECD) 38 to obtain sequence information. ECD has been demonstrated to be especially valuable for mapping phosphorylation sites, because it preserves the labile PTMs during MS/MS process.…”

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy

“…Electron capture dissociation (ECD), for example, has been shown to achieve effective cleavage of large polypeptides and small proteins [8]. This method induces peptide fragmentation, primarily at N-C␣ bonds, using low-energy electrons [9]. Discussion continues as to the precise nature of the fragmentation mechanisms induced by ECD [10,11,12], but clearly the radical-induced fragmentations occur at low internal energy and are qualitatively different to those induced by CAD [13].…”

“…For instance, facile neutral loss of phosphoric acid, as commonly observed in CAD spectra of phosphopeptides, is rarely observed using ECD. This has prompted a strong focus on the use of ECD for post-translational modification analysis [14], but ECD has also proven successful more generally in generating peptide and protein identification data [8,9,13,15,16]. The implementation of ECD is however largely restricted to ion cyclotron resonance instruments.…”