Electron capture dissociation of highly charged proteolytic peptides from Lys N, Lys C and Glu C digestion

Abstract

Electron capture dissociation (ECD), which provides more extensive sequence coverage compared to “slow heating” tandem mass spectrometric techniques such as collision-activated dissociation, constitutes a promising method for de novo sequencing of peptides and proteins. We have previously examined and characterized the ECD fragmentation behavior of small to medium size doubly and triply protonated peptides from trypsin, chymotrypsin and Glu C digestion. Here, we extend that work to longer, more highly charged proteolytic peptides from Lys N, Lys C and Glu C digestion to compare the extent of fragmentation obtained from each type of proteolytically derived peptide and also to examine the effectiveness of each enzyme in terms of peptide sequence coverage in ECD. Our findings demonstrate that medium size peptides (i.e., 1600–4800 Da) at high charge states (+3 to +6) exhibit very similar ECD fragmentation behavior independent of which proteolytic enzyme was used for digestion. The average peptide sequence coverage we obtained for Lys N and Glu C generated peptides was 81%, and for Lys C proteolytic peptides we obtained an average sequence coverage of 80%. Most of the peptides we examined, 82%, showed high ECD sequence coverage ranging from 70–100%, whereas 13% of the peptides showed a moderate sequence coverage ranging from 50–60%. Only 5% of the peptides showed an average sequence coverage below 50%. Furthermore, the extent of fragmentation, measured by the total number of backbone c- and z-type product ions, was very similar for Lys N, Lys C and Glu C derived peptides.