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.
The novel technique electron capture dissociation (ECD) of electrospray generated [M + nH]n+ polypeptide cations produces rapid cleavage of the backbone NH-Ca bond to form c and z ions (in the modified notation of Roepstorff and Fohlman). The potential of the Fourier transform mass spectrometry equipped with ECD in structure analysis of O-glycosylated peptides in the 3 kDa range has been investigated. Totally, 85% of the available interresidue bonds were cleaved in five glycopeptides; more stable c ions accounted for 62% of the observed fragmentation. The c series provided direct evidence on the glycosylation sites in every case studied, with no glycan (GalNAc and dimannose) losses observed from these species. Less stable z ions supported the glycan site assignment, with minor glycan detachments. These losses, as well as the observed formation of even-electron z ions, are attributed to radical-site-initiated reactions. In favorable cases, complete sequence and glycan position information is obtained from a single-scan spectrum. The "mild" character of ECD supports the previously proposed non-ergodic (cleavage prior to energy randomization) mechanism, and the low internal energy increment of fragments.
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Reactions of electrons in the energy range below 70 eV with polypeptide cations and anions are reviewed, as well as their applications for the structural analysis of polypeptides. At very low energies (= 0.1 eV), the major outcome is electron-capture dissociation (ECD) of S-S and backbone N-C(alpha) bonds, leading to c' and z. fragments. ECD is useful in sequencing and characterization of post-translational modifications (PTMs), because c', z. fragmentation is abundant and the fragments usually retain labile groups. Electron capture at higher energies (3-13 eV) induces secondary fragmentation in radical z. fragments; this hot ECD (HECD) allows one to distinguish between the isomeric leucine and isoleucine residues. If a hot electron is not captured, then the induced electronic excitation converts internally into vibrational energy, resulting in fragmentation of the C(O)bond;N backbone bond (so-called EIEIO process). Above 9-10 eV, further ionization of n-charged cations occurs. If the formed (n + 1)+. cations capture electrons, then the C(alpha)bond;C backbone bond is usually broken. For anions that collide with approximately 20 eV electrons, the ejection of an electron leads to the creation of a radical positive charge (hole) that recombines internally with a negative charge. Such recombination leads to various backbone bond cleavages. This electron-detachment dissociation (EDD) is analogous to ECD for negative ions.
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