Studies of Peptide a- and b-Type Fragment Ions Using Stable Isotope Labeling and Integrated Ion Mobility/Tandem Mass Spectrometry

The structures of peptide a- and b-type fragment ions were studied using synthetic peptides including a set of isomeric peptides, differing in the sequence location of an alanine residue labeled with 15N and uniformly with 13C. The pattern of isotope labeling of second-generation fragment ions derived via an and bn ions (where n = 4 or 5) suggested that these intermediates existed in part as macrocyclic structures, where alternative sites of ring opening gave rise to different linear forms whose simple cleavage might give rise to the observed final products. Similar conclusions were derived from combined ion mobility/tandem MS analyses where different fragmentation patterns were observed for isomeric a- or b-type ions that display different ion mobilities. These analyses were facilitated by a new approach to the processing of ion mobility/tandem MS data, from which distinct and separate product ion spectra are derived from ions that are incompletely separated by ion mobility. Finally, an example is provided of evidence for a macrocyclic structure for bn ions where n = 8 or 9.

Graphical AbstractThe use of stable isotope labeling and combined ion mobility/tandem MS provides additional insights into the contribution of macrocyclic intermediates in peptide ion fragmentation.Figure optionsDownload full-size imageDownload high-quality image (118 K)Download as PowerPoint slide