The fragmentation mechanisms of singly protonated Gly-Asp-Gly-Arg (GDGRI and Arg-Gly-Asp-Gly (RGDGJ 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 (Qc + QN) 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 (Qc + QN] for the singly charged peptides tended to be negative when the proton was located at the backbone nitrogen atoms. A relatively larger value of QC + QN 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 exother- mic, 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.