Electron Capture Dissociation for Structural Characterization of Multiply Charged Protein Cations

TLDR

For proteins under 20 kDa, electron capture dissociation cleaves many more backbone bonds than conventional collisionally activated dissociation, with capture efficiency maximized by a minimal kinetic energy difference between electron and ion. In an FTMS cell equipped with electron‑trapping electrodes, capture occurs at the boundary of the electron and ion potential wells, yielding ~80 % precursor conversion, with a z²‑dependent cross section that favors nonergodic dissociation via protonated sites and high‑affinity H• capture. ECD produced complete backbone cleavages for mellitin and ubiquitin, enabling de novo sequencing, and preserved post‑translational modifications better than CAD, yielding more specific sequence assignments.

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 103. 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 (∼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.