Side-chain losses in electron capture dissociation to improve peptide identification

Analysis of a database of some 20 000 conventional electron-capture dissociation (ECD) mass spectra of doubly charged ions belonging to tryptic peptides revealed widespread appearance of w ions and related u ions that are due to partial side chain losses from radical z(center dot) ions. Half of all z(center dot) ions that begin with Leu or Ile produce w ions in conventional one-scan ECD mass spectra, which differentiates these isomeric residues with > 97% reliability. Other residues exhibiting equally frequent side chain losses are Gln, Glu, Asp, and Met (cysteine was not included in this work). Unexpectedly, Asp lost not a radical group like other amino acids but a molecule CO2, thus giving rise to a radical w(center dot) ion with the possibility of a radical cascade. Losses from amino acids as distant as seven residues away from the cleavage site were detected. The mechanism of such losses seems to be related to radical migration from the original site at the C-alpha(n) atom in a z(n)center dot ion to other C-alpha and C-beta atoms. The side chain losses confirm sequence assignment, improve the database matching score, and can be useful in de novo sequencing.