Comparison of the electron capture dissociation fragmentation behavior of doubly and triply protonated peptides from trypsin, Glu-C, and chymotrypsin digestion.

In bottom-up proteomics, proteolytically derived peptides from proteins of interest are analyzed to provide sequence information for protein identification and characterization. Electron capture dissociation (ECD), which provides more random cleavages compared to "slow heating" techniques such as collisional activation, can result in greater sequence coverage for peptides and proteins. Most bottom-up proteomics approaches rely on tryptic doubly protonated peptides for generating sequence information. However, the effectiveness, in terms of peptide sequence coverage, of tryptic doubly protonated peptides in ECD remains to be characterized. Herein, we examine the ECD fragmentation behavior of 64 doubly- and 64 triply protonated peptides (i.e., a total of 128 peptide ions) from trypsin, Glu-C, and chymotrypsin digestion in a Fourier transform ion cyclotron resonance mass spectrometer. Our findings indicate that when triply protonated peptides are fragmented in ECD, independent of which proteolytic enzyme was used for protein digestion, more c- and z-type product ions are observed, and the number of complementary fragment pairs increases dramatically (44%). In addition, triply protonated peptides provide an increase (26%) in peptide sequence coverage. ECD of tryptic peptides, in both charge states, resulted in higher sequence coverage compared to chymotryptic and Glu-C digest peptides. The peptide sequence coverage we obtained in ECD of tryptic doubly protonated peptides (64%) is very similar to that reported for electron transfer dissociation of the same peptide type (63%).