Replacing H by Na or K in phosphopeptide anions and cations prevents electron capture dissociation.

By successively replacing H by Na or K in phosphopeptide anions and cations, we show that the efficiency of fragmentation into and ˙ or ˙ and fragments from N-Cα backbone bond cleavage by negative ion electron capture dissociation (niECD) and electron capture dissociation (ECD) substantially decreases with increasing number of alkali ions attached. In proton-deficient phosphopeptide ions with a net charge of 2-, we observed an exponential decrease in electron capture efficiency with increasing number of Na or K ions attached, suggesting that electrons are preferentially captured at protonated sites. In proton-abundant phosphopeptide ions with a net charge of 3+, the electron capture efficiency was not affected by replacing up to four H ions with Na or K ions, but the yield of , ˙ and ˙, fragments from N-Cα backbone bond cleavage generally decreased next to Na or K binding sites. We interpret the site-specific decrease in fragmentation efficiency as Na or K binding to backbone amide oxygen in competition with interactions of protonated sites that would otherwise lead to backbone cleavage into , ˙ or ˙, fragments. Our findings seriously challenge the hypothesis that the positive charge responsible for ECD into , ˙ or ˙, fragments can generally be a sodium or other metal ion instead of a proton.