Comparative Studies on the Discrepant Fragmentation Mechanisms of the Gly-Asp-Gly-Arg and Arg-Gly-Asp-Gly: Evidence for the Mobile Proton Model

The fragmentation mechanisms of singly protonated Gly-Asp-Gly-Arg (GDGR) and Arg-Gly-Asp-Gly (RGDG) were investigated by mass spectrometry and theoretical methods. Both protonated molecules are fragmented mainly at the Asp–Gly amide bond C-terminal to Asp, as supported by quantum chemical calculations. Charge distributions of C and N atoms (Q_C + Q_N) on the amide bonds were collected when the ionizing proton was fixed at different nitrogen atoms along the backbone for each peptide. Compared with the neutral molecules, the total charges of C and N atoms (Q_C + Q_N) for the singly charged peptides tended to be negative when the proton was located at the backbone nitrogen atoms. A relatively larger value of Q_C + Q_N corresponds to a higher trend of fragmentation, which is consistent with the experimental relative abundances data that the predominant ions are y₂ for [GDGR + H]⁺ and b₃ for [RGDG + H]⁺. Also, the anhydride mechanism driven by the C-terminal COOH for [RGDG + H]⁺ was explored by a quantum–mechanical/molecular–mechanical method. Calculations indicate that the protonated peptide can be cleaved through an unusual charge-directed pathway by forming a salt bridge at the C-termini. The formation of the anhydride linkage is much more feasible since this process needs very little energy and is exothermic, though the subsequent nucleophilic attack on the Asp carbonyl carbon is more difficult. The combined experimental and theoretical methods substantiate the mobile proton model, which opens a way to analyze quantitatively the discrepant fragmentation of dissociated peptides in peptide/protein identification.