The relative charge ratio between C and N atoms in amide bond acts as a key factor to determine peptide fragment efficiency in different charge states.

The influence of charge state on the peptide dissociation behavior in tandem mass spectrometry (MS/MS) is worthy of discussion. Comparative studies of singly- and doubly-protonated peptide molecules are performed to explore the effect and mechanism of charge state on peptide fragmentation. In view of the charge-directed cleavage of protonated peptides described in the mobile proton model, radiolytic oxidation was applied to change the charge distribution of peptides but retain the sequence. Experimental studies of collision energy-dependent fragmentation efficiencies coupled with quantum chemical calculations indicated that the cleavage of ARRA and its side-chain oxidation products with oxygen atoms added followed a trend that doubly-protonated peptides fragment more easily than singly-protonated forms, while the oxidation product with the guanidine group deleted showed the opposite trend. By analyzing the charge distribution around the amide bonds, we found that the relative charge ratios between C and N atoms (Q(C)/Q(N)) in the amide bonds provided a reasonable explanation for peptide fragmentation efficiencies. An increase of the Q(C)/Q(N) value of the amide bond means that a peptide fragments more easily, and vice versa. The results described in this paper provide an experimental and calculation strategy for predicting peptide fragmentation efficiency.