Activated ion negative electron transfer dissociation of multiply charged peptide anions

We report the implementation and evaluation of activated ion negative electron transfer dissociation (AI-NETD) in order to enhance the analytical capabilities of NETD for the elucidation of doubly deprotonated peptide anions. The analytical figures-of-merit and fragmentation characteristics are compared for NETD alone and with supplemental collisional activation of the charge reduced precursors or infrared photoactivation of the entire ion population during the NETD reaction period. The addition of supplemental collisional activation of charge reduced precursor ions or infrared photoactivation of the entire ion population concomitant with the NETD reaction period significantly improves sequencing capabilities for peptide anions as evidenced by the greater abundances of product ions and overall sequence coverage. Neither of these two AI-NETD methods significantly alters the net fragmentation efficiencies relative to NETD; however, the sequence ion conversion percentages with respect to formation of diagnostic product ions are notably higher. Supplemental infrared photoactivation outperforms collisional activation for most of the peptide fragmentation metrics evaluated.