Fragmentation of Protonated Tripeptides:  The Proline Effect Revisited

The fragmentation of protonated tripeptides under metastable ion conditions and collision-induced conditions are reported. The majority of protonated tripeptides cleave at the C-terminal amide bond to form b 2 and y 1 ions, with the relative amounts depending on the proton affinities of the amino acids derived from the C-terminal amino acid residue. Protonated tripeptides that have a proline residue at the C-terminal position fragment almost exclusively by cleavage of the amide bond adjacent to the proline and give mainly y 1 ions; where there is a proline residue in the central position, fragmentation of the protonated tripeptide occurs at both the N-terminal and the C-terminal amide bonds to form y 2 and b 2 ions; finally, two of the tripeptides with proline at the N-terminal position, Pro-Pro-Pro and Pro-Gly-Gly, fragment largely by cleavage at the N-terminal, the former by cleavage of the amide bond and the latter by direct formation of the a 1 ion. Protonated Pro-Val-Gly fragments to give predominantly b 2 and a 2 ions at low energy, but the a 1 ion becomes the dominant product at higher energies. In the fragmentation of protonated peptides, the lack of b ions formed by cleavage at the amide bond C-terminal to proline (i.e., with the proline residue in the oxazolone ring) has conventionally been attributed to ring strain in a bicyclic oxazolone. Here we show, however, that the bicyclic oxazolone structure containing proline derived from protonated Gly-Pro-Gly is only 2.7 kcal/mol higher in free energy, at the B3LYP/6-31++G(d,p) level of theory, than the isomeric nonbicyclic oxazolone derived from protonated Pro−Gly−Gly; that is, strain appears to be a small or negligible factor. Formation of the y 2 ion in protonated Gly-Pro-Gly has a barrier of 3 kcal/mol higher free energy than that of the b 2 ion. In comparison, the difference increases to 10 kcal/mol in protonated Gly-Phe-Gly. The neutral products that are formed along with the y 2 ion are CO and methanimine, not aziridinone as once proposed.