The cleavage processes of protonated peptides in mass spectrometry, described in the mobile proton model, are charge-directed and depend on the charge distribution around the cleavage sites. Previous studies experimentally verified the mobile proton model by changing peptide sequences. In this study, oxidation was applied to change the charge distribution of peptides, but the sequence was retained. Tandem mass spectrometry (MS/MS) and quantum chemical calculations at the B3LYP/6-31G(d) level were used to test the validity of the mobile proton model. The results showed prominent differences of peptide fragmentation efficiency caused by the charge distribution produced by various oxidation levels. Fragmentation efficiency curves coupled with the relative intensities of the fragments indicated that the cleavage of the peptide Ala-Arg-Arg-Ala (ARRA) became more and more difficult as 0 atoms were added. The relative charge ratios between C and N atoms in the amide bonds decreased with the increase of oxidation extent, suggesting that oxidation resulted in protons moving away from the amide bonds. The combined methods proposed here provide a unique approach to substantiate and refine the mobile proton model for peptide fragmentation.