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 c and z˙ or c˙ and z 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 c, z˙ and c˙, z 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 c, z˙ or c˙, z fragments. Our findings seriously challenge the hypothesis that the positive charge responsible for ECD into c, z˙ or c˙, z fragments can generally be a sodium or other metal ion instead of a proton.