Gas Phase Fragmentation Behavior of Proline in Macrocyclic b₇ Ions

The fragmentation characteristics of b₇ ions produced from proline-containing heptapeptides have been studied in detail. The study has utilized the following C-terminally amidated model peptides: PA₆, APA₅, A₂PA₄, A₃PA₃, A₄PA₂, A₅PA, A₆P, PYAGFLV, PAGFLVY, PGFLVYA, PFLVYAG, PLVYAGF, PVYAGFL, YPAGFLV, YAPGFLV, YAGPFLV, YAGFPLV, YAGFLPV, YAGFLVP, PYAFLVG, PVLFYAG, A₂PXA₃, and A₂XPA₃ (where X = C, D, F, G, L, V, and Y, respectively). The results have shown that b₇ ions undergo head-to-tail cyclization and form a macrocyclic structure. Under the collision-induced dissociation (CID) condition, it generates nondirect sequence ions regardless of the position of the proline and the neighboring amino acid residues. This study highlights the unusual and unique fragmentation behavior of proline-containing heptapeptides. Following the head-to-tail cyclization, the ring opens up and places the proline residue in the N-terminal position while forming a regular oxazolone form of b₂ ions for all peptide series. Then, the fragmentation reaction pathway is followed by the elimination of proline with its C-terminal neighbor residue as an oxazolone (e.g., PX_oxa) for all proline-containing peptide series.