In this study, we investigated the effect of the second amino acid identity of hexapeptides on gas-phase structures and the proton affinities of N-terminal proline containing b(2)(+) ions produced from the fragmentation of b(6)(+) ions under low-energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). It should be noted that, among all other fragments, the b(2)(+) and nominally b(4)(+) (AAAA) ions ([M+H](+) -> b(G)(+) -> b(2)(+) (PX+)+ b(4)(+) (AAAA(+)) were mainly considered in this study. This is a unique example of consecutive cleavage of b6+ ions which fragments to b2+ and nominal b(4)(+) ions. All structural and proton affinity calculations for b2+ ions were carried out with the B3LYP/6-31+G(d,p) level of theory. The study utilized C-terminal amidated model peptides consisting of PAAAAA-NH2 and PXAAAA-NH2 where Xis phenylalanine (F), glutamic acid (E), tryptophan (W), and histidine (H) residue. Two main structural isomers of b(2)(+) ions, namely oxazolone and diketopiperazine, have been considered for the computations. The results demonstrated that the proton affinities of oxazolone isomers of PX are greater than its diketopiperazine isomers. Higher correlation coefficient is calculated if the structure of PX is considered as oxazolone rather than diketopiperazine isomer. Additionally, a linear fit is observed between intensity ratio (PX/AAAA) and calculated proton affinities of PX ions. Additionally, MS/MS results revealed that the relative intensities of b(2)(+)-PA, PF, and PE- ions are lower compared to the relative intensity of AAAA fragment ion. In contrast, b(2)(+)-PW and PH- ions have higher relative intensities compared to the AAAA ion. This behavior is explained by the proton affinities of fragment ions computationally. (C) 2015 Elsevier B.V. All rights reserved.