When ionized by electrospray from acidic solutions, the tripeptides Pro-His-Xaa (Xaa = Gly, Ala, Leu) form abundant doubly-protonated ions, [M + 2H]2+. Collision-induced dissociation (CID) of these doubly-protonated species results, in part, in formation of b(2)(2+) ions, which fragment further by loss of CO to form a(2)(2+) ions; the latter fragment by loss of CO to form the Pro and His iminium [immonium is commonly used in peptide MS work] ions. Although larger doubly-charged b ions are known, this represents the first detailed study of b(2)(2+) ions in CID of small doubly protonated peptides. The most abundant CID products of the studied doubly-protonated peptides arise mainly in charge separation involving two primary fragmentation channels, formation of the b2/y1 pair and formation of the a1/y2 pair. Combined molecular dynamics and density functional theory calculations are used to gain insight into the structures and fragmentation pathways of doubly-protonated Pro-His-Gly including the energetics of potential protonation sites, backbone cleavages, post-cleavage charge-separation reactions and the isomeric structures of b(2)(2+) ions. Three possible structures are considered for the b(2)(2+) ions: the oxazolone, diketopiperazine, and fused ring isomers. The last is formed by cleavage of the His-Gly amide bond on a pathway that is initiated by nucleophilic attack of one of the His side-chain imidazole nitrogens. Our calculations indicate the b(2)(2+) ion population is dominated by the oxazolone and/or fused ring isomers.