Ion activation in electron capture dissociation to distinguish between N-terminal and C-terminal product ions

We present a method to distinguish N-terminal from C-terminal product ions in electron capture dissociation (ECD) MS/MS due to the change in relative abundances of even-electron (prime) and odd-electron (radical) product ions produced in consecutive ECD and activated ion-ECD mass spectra. The method is based on the rate and direction of hydrogen atom transfer between N-terminal and C-terminal ECD products and its dependence on ion internal energy. We demonstrate that increasing ion internal energy by vibrational activation prior to ECD results in decreased ratio of radical/prime N-terminal product ions (c^./c′ ratio), but increased ratio of radical/prime C-terminal product ions (z^./z′ ratio) in many cases. The combination of AI-ECD and ECD promises to increase the confidence of mass spectrometry-based peptide sequencing and protein identification.